Physiological and developmental dysfunctions in the dengue vector Culex pipiens (Diptera: Culicidae) immature stages following treatment with zinc oxide nanoparticles.

The medical value of mosquitoes attracted researchers worldwide to search for a valuable way to control such serious insects. The continuous development of resistance against chemical insecticides pushed toward looking for novel and promising compounds against mosquitoes. In this study, the toxicity and physio-developmental effects of 10-30 nm spherical zinc oxide nanoparticles (ZnONPs) in aqueous suspension was addressed against the first larval instar of Culex pipiens mosquito. The calculated value of LC50 was about 0.892 g/L while the sub lethal concentration LC20 recorded about 0.246 g/L. Larvae treated with ZnONPs suffered reduced growth rate, longer developmental period and malformations in the breathing tube. Furthermore, the treated larvae showed clear abnormal appearance of the gastric caeca and midgut epithelia under transmission electron microscope (TEM). These abnormalities appeared as condensation of the nuclear chromatin, abnormal shape or absence of microvilli, highly increased amount of smooth endoplasmic reticulum in the cytoplasm and appearance of numerous vacuoles. Additionally, ZnONPs interfered with several biochemical pathways such as induction of oxidative stress which appeared in the form of increased levels of hydrogen peroxide and inability to activate the detoxifying enzymes alkaline phosphatase (ALP), catalase and glutathione peroxidase (GPX). On the contrary, the activity of the antioxidant enzyme superoxide dismutase (SOD) increased in treated larvae. Furthermore, LC20 and LC50 of ZnONPs inhibited the growth rate of the larval gut fauna in vitro. These results clearly show that ZnONPs target several tissues leading to serious alteration in the physiological and developmental processes in C. pipiens mosquito larvae.

Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses.

Several immunosuppressive factors are associated with parasitism of an endoparasitoid wasp, Cotesia plutellae, on the diamondback moth, Plutella xylostella. C. plutellae bracovirus (CpBV) encodes a large number of putative protein tyrosine phosphatases (PTPs), which may play a role in inhibiting host cellular immunity. To address this inhibitory hypothesis of CpBV-PTPs, we performed transient expression of individual CpBV-PTPs in hemocytes of the beet armyworm, Spodoptera exigua, and analyzed their cellular immune responses. Two different forms of CpBV-PTPs were chosen and cloned into a eukaryotic expression vector under the control of the p10 promoter of baculovirus: one with the normal cysteine active site (CpBV-PTP1) and the other with a mutated active site (CpBV-PTP5). The hemocytes transfected with CpBV-PTP1 significantly increased in PTP activity compared to control hemocytes, but those with CpBV-PTP5 exhibited a significant decrease in the PTP activity. All transfected hemocytes exhibited a significant reduction in both cell spreading and encapsulation activities compared to control hemocytes. Co-transfection of CpBV-PTP1 together with its double-stranded RNA reduced the messenger RNA (mRNA) level of CpBV-PTP1 and resulted in recovery of both hemocyte behaviors. This is the first report demonstrating that the polydnaviral PTPs can manipulate PTP activity of the hemocytes to interrupt cellular immune responses.

Protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus: sequence analysis, expression profile, and a possible biological role in host immunosuppression.

A genome project has been launched and aims to sequence total genome of Cotesia plutellae bracovirus (CpBV). On this process, several putative open reading frames have been proposed, among which there was a large gene family coding for protein tyrosine phosphatases (PTPs). This study analyzed the deduced amino acid sequences of 14 CpBV-PTPs in terms of conserved domains with other known polydnaviral PTPs and determined their expression patterns in diamondback moth, Plutella xylostella, parasitized by C. plutellae. The analyzed CpBV-PTPs share the common 10 motifs with classical type of PTPs. However, there are variations among CpBV-PTPs in active site sequence and phosphorylation sites. Quantitative real-time polymerase chain reaction (PCR) indicated that most PTPs in the parasitized P. xylostella were expressed from the first day of parasitization and increased the expression levels during parasitization. All 14 PTPs were expressed in both immune-associated tissues of fat body and hemocytes in the parasitized host. During last instar, the PTP enzyme activity of the parasitized P. xylostella was significantly lower than that of the nonparasitized. The reduction of the PTP activity was observed in cytosolic fraction, but not in membrane fraction. The hemocytes of parasitized P. xylostella markedly lost their spreading ability in response to a cytokine (PSP1: plasmatocyte-spreading peptide 1). The functional link between the reduced PTP activity and the suppressed hemocytic behavior was evidenced by the inhibitory effect of sodium orthovanadate (a specific PTP inhibitor) on hemocyte-spreading behavior of nonparasitized P. xylostella. These results suggest that CpBV-PTPs are expressed in the parasitized P. xylostella and affect cellular PTP activity, which may be associated with host immunosuppression.

Parasitism by Cotesia plutellae alters the hemocyte population and immunological function of the diamondback moth, Plutella xylostella.

Cotesia plutellae, a solitary endoparasitoid wasp, parasitizes the diamondback moth, Plutella xylostella, and induces host immunosuppression and lethality in the late larval stage. This study focused on changes of cellular immunity in the parasitized P. xylostella in terms of hemocyte composition and cellular functions. In third and fourth instar larvae of nonparasitized P. xylostella, granular cells represented the main hemocyte type (60-70%) and plasmatocytes were also present at around 15% among the total hemocytes. Following parasitization by C. plutellae, the relative proportions of these two major hemocytes changed very little, but the total hemocyte counts exhibited a significant reduction. Functionally, the granular cells played a significant role in phagocytosis based on a fluorescence assay using fluorecein isothiocyanate-labeled bacteria. The phagocytic activity of the granular cells occurred as early as 5 min after incubation with the bacteria, and increased during the first 40 min of incubation. The parasitism by C. plutellae significantly inhibited phagocytosis of the granular cells. Plasmatocytes also exhibited minor phagocytic activity. Moreover, plasmatocyte phagocytosis was not inhibited by parasitism. On the other hand, hemocyte-spreading behavior in response to pathogen infection was significant only for plasmatocytes, which exhibited a characteristic spindle shape upon infection. A significant spreading of the plasmatocytes was found as early as 5 min after pathogen incubation and their ratio increased during the first 40 min. An insect cytokine, plasmatocyte-spreading peptide 1 (PSP1) from Pseudoplusia includens, was highly active in inducing plasmatocyte-spreading behavior of P. xylostella in a dose-dependent manner. P. xylostella parasitized by C. plutella was significantly inhibited in plasmatocyte-spreading in response to an active dose of PSP1. An in vivo encapsulation assay showed that the parasitized P. xylostella could not effectively form the hemocyte capsules around injected agarose beads. This research demonstrates that the parasitism of C. plutellae adversely affects the total hemocyte populations in number and function, which would contribute to host immunosuppression.