Insect Salivary Glands Research Articles

Viral and cellular determinants of polarized trafficking of viral envelope proteins from insect-specific and insect-vectored viruses in insect midgut and salivary gland cells.

Viruses that use insects as hosts must navigate specific routes through different insect tissues to complete their life cycles. The routes may differ substantially depending on the life cycle of the virus. Both insect pathogenic viruses and insect-vectored viruses must navigate through the polarized cells of the midgut epithelium to establish a systemic infection. In addition, insect-vectored viruses must also navigate through the polarized salivary gland epithelium for transmission. Thus, insect-vectored viruses appear to traffic in opposite directions in these two tissues. In this study, we asked whether two viral envelope proteins (VSV G and baculovirus GP64) alone encode the signals necessary for the polarized trafficking associated with their respective life cycles. Using Drosophila as a model to examine tissue-specific polarized trafficking of these viral envelope proteins, we identified one of the virus-encoded signals and several host proteins associated with regulating the polarized trafficking in the midgut epithelium.

Scavenging H2O2 of plant host by saliva catalase of leafhopper vector benefits viral transmission.

Catalase (CAT) is the main reactive oxygen species (ROS)-scavenging enzyme in plants and insects. However, it remains elusive whether and how insect saliva CAT suppresses ROS-mediated plant defense, thereby promoting initial virus transmission by insect vectors. Here, we investigated how leafhopper Recilia dorsalis catalase (RdCAT) was secreted from insect salivary glands into rice phloem, and how it was perceived by rice chaperone NO CATALASE ACTIVITY1 (OsNCA1) to scavenge excessive H2O2 during insect-to-plant virus transmission. We found that the interaction of OsNCA1 with RdCAT activated its enzymatic activity to decompose H2O2 in rice plants during leafhopper feeding. However, initial insect feeding did not significantly change rice CATs transcripts. Knockout of OsNCA1 in transgenic lines decreased leafhopper feeding-activated CAT activity and caused higher H2O2 accumulation. A devastating rice reovirus activated RdCAT expression and promoted the cosecretion of virions and RdCAT into leafhopper salivary cavities and ultimately into the phloem. Virus-mediated increase of RdCAT secretion suppressed excessive H2O2, thereby promoting host attractiveness to insect vectors and initial virus transmission. Our findings provide insights into how insect saliva CAT is secreted and perceived by plant chaperones to suppress the early H2O2 burst during insect feeding, thereby facilitating viral transmission.

Nitric oxide delivery and heme-assisted S-nitrosation by the bedbug nitrophorin

Nitrophorins are heme proteins used by blood feeding insects to deliver nitric oxide (NO) to a victim, leading to vasodilation and antiplatelet activity. Cimex lectularius (bedbug) nitrophorin (cNP) accomplishes this with a cysteine ligated ferric (Fe(III)) heme. In the acidic environment of the insect's salivary glands, NO binds tightly to cNP. During a blood meal, cNP-NO is delivered to the feeding site where dilution and increased pH lead to NO release. In a previous study, cNP was shown to not only bind heme, but to also nitrosate the proximal cysteine, leading to Cys-NO (SNO) formation. SNO formation requires oxidation of the proximal cysteine, which was proposed to be metal-assisted through accompanying reduction of ferric heme and formation of Fe(II)-NO. Here, we report the 1.6 Å crystal structure of cNP first chemically reduced and then exposed to NO, and show that Fe(II)-NO is formed but SNO is not, supporting a metal-assisted SNO formation mechanism. Crystallographic and spectroscopic studies of mutated cNP show that steric crowding of the proximal site inhibits SNO formation while a sterically relaxed proximal site enhances SNO formation, providing insight into specificity for this poorly understood modification. Experiments examining the pH dependence for NO implicate direct protonation of the proximal cysteine as the underlying mechanism. At lower pH, thiol heme ligation predominates, leading to a smaller trans effect and 60-fold enhanced NO affinity (Kd = 70 nM). Unexpectedly, we find that thiol formation interferes with SNO formation, suggesting cNP-SNO is unlikely to form in the insect salivary glands.

Knockdown of the salivary protein gene NlG14 caused displacement of the lateral oviduct secreted components and inhibited ovulation in Nilaparvata lugens.

Saliva plays important roles in insect feeding, but its roles in insect reproduction were rarely reported. Here we reported that the knockdown of a salivary gland-specific gene NlG14 disrupted the reproduction through inhibiting the ovulation of the brown planthopper (BPH), Nilaparvata lugens (Stål), one of the most devastating rice pests in Asia. NlG14 knockdown caused the displacement of the lateral oviduct secreted components (LOSC), leading to the ovulation disorder and the accumulation of mature eggs in the ovary. The RNAi-treated females laid much less eggs than their control counterparts, though they had the similar oviposition behavior on rice stems as controls. NlG14 protein was not secreted into the hemolymph, indicating an indirect effect of NlG14 knockdown on BPH reproduction. NlG14 knockdown caused the malformation of A-follicle of the principal gland and affected the underlying endocrine mechanism of salivary glands. NlG14 reduction might promote the secretion of insulin-like peptides NlILP1 and NlILP3 from the brain, which up-regulated the expression of Nllaminin gene and then caused the abnormal contraction of lateral oviduct muscle. Another explanation was NlG14 reduction disrupted the ecdysone biosynthesis and action through the insulin-PI3K-Akt signaling in ovary. Altogether, this study indicated that the salivary gland specific protein NlG14 indirectly mediated BPH ovulation process, which established a connexon in function between insect salivary gland and ovary.

Competence of mosquitoes <i>Culex pipiens f.</i> molestus as carriers of West Nile virus under various temperature conditions

Introduction. The Culex pipiens mosquito is one of the proven vectors of the West Nile virus (WNV). Culex pipiens f. molestus (Cx. p. f. molestus) is a synanthropic, autogenous, widespread form of the species that can feed on a broad range of hosts, including humans. The temperature of the habitat of insects affects the potential for virus transmission, which determines the likelihood of them carrying the pathogen of West Nile fever.
 The goal is an experimental study of the temperature of the habitat of larvae on the competence of mosquitoes Cx. p. f. molestus as carriers of WNV.
 Materials and methods. We used a strain of the WNV (WNV_Volg601/18 genotype 2) and a laboratory culture of mosquitoes Cx. p. f. molestus. The concentration of the virus was detected by plaque formation using Vero cells. Insects were infected orally at the larval stage, with subsequent incubation at 20, 22 or 28C. 72 hours after the emergence of all adults from the pupae, the mosquitoes were immobilized by cold, the sex of imago was determined, the salivary glands were isolated from the females, and the presence of WNV in glandes and its titer were detected.
 Results. The titer of WNV sufficient to transmit the pathogen through the insect biting was observed in the salivary glands of insects kept at a temperature of 22 and 28C, with the virus titer rising with the temperature increasing. No virus was detected in the salivary glands of female insects kept at a temperature of 20C.
 Conclusion. Thus, it appears that the habitat temperature is an important factor limiting the replication and content of WNV in the salivary glands of Cx. p. f. molestus.

Catalyzing systemic movement of inward rectifier potassium channel inhibitors for antifeedant activity against the cotton aphid, Aphis gossypii (Glover).

The cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is a destructive agricultural pest, capable of photosynthate removal and plant virus transmission. Therefore, we aimed to test the antifeedant properties of small-molecule inhibitors of inward rectifier potassium (Kir) channels expressed in insect salivary glands and develop an approach for enabling systemic movement of lipophilic Kir inhibitors. Two Kir channel inhibitors, VU041 and VU730, reduced the secretory activity of the aphid salivary glands by 3.3-fold and foliar applications of VU041 and VU730 significantly (P < 0.05) increased the time to first probe, total probe duration, and nearly eliminated phloem salivation and ingestion. Next, we aimed to facilitate systemic movement of VU041 and VU730 through evaluation of a novel natural product based solubilizer containing rubusoside that was isolated from Chinese sweet leaf (Rubus suavissimus) plants. A single lower leaf was treated with Kir inhibitor soluble liquid (KI-SL) and systemic movement throughout the plant was verified via toxicity bioassays and changes to feeding behavior through the electrical penetration graph (EPG) technique. EPG data indicate KI-SL significantly reduced ability to reach E1 (phloem salivation) and E2 (phloem ingestion) waveforms and altered plant probing behavior when compared to the untreated control. High-performance liquid chromatography (HPLC) analysis indicated the presence of VU041 and VU730 in the upper leaf tissue of these plants. Together, these data provide strong support that incorporation of rubusoside with Kir inhibitors enhanced translaminar and translocation movement through the plant tissue. These data further support hemipteran Kir channels as a target to prevent feeding and induce toxicity. Further, these studies highlight a novel delivery approach for generating plant systemic activity of lipophilic insecticides. © 2022 Society of Chemical Industry.

Key role of exportin 6 in exosome-mediated viral transmission from insect vectors to plants

Exosomes play a key role in virus exocytosis and transmission. The exportin family is usually responsible for cargo nucleocytoplasmic trafficking, and they are frequently found in exosomes. However, the function of exportins sorted in exosomes remains unknown. Here, we successfully isolated "cup holder"-like exosomes from the saliva of ∼30,000 small brown planthoppers, which are vectors of rice stripe virus (RSV). RSV virions were packed in comparatively large exosomes. Four viral genomic RNAs at a certain ratio were identified in the saliva exosomes. The virions contained in the saliva exosomes were capable of replicating and causing disease in rice plants. Interference with each phase of the insect exosome system affected the transmission of RSV from the insect vectors to rice plants. Fragmented exportin 6 was coimmunoprecipitated with viral nucleocapsid protein in saliva and sorted to exosomes via interactions with the cargo sorting protein VPS37a. When the expression of exportin 6 was knocked down, the amounts of RSV secreted in saliva and rice plants were reduced by 60% and 74%, respectively. These results showed that exportin 6 acted as a vehicle for transporting RSV into exosomes to overcome the barrier of insect salivary glands for horizontal transmission. Exportin 6 would represent an ideal target that could be manipulated to control the outbreak of insect-borne viruses in the future.

A salivary gland-secreted peptide regulates insect systemic growth.

Insect salivary glands have been previously shown to function in pupal attachment and food lubrication by secreting factors into the lumen via an exocrine way. Here, we find in Drosophila that a salivary gland-derived secreted factor (Sgsf) peptide regulates systemic growth via an endocrine way. Sgsf is specifically expressed in salivary glands and secreted into the hemolymph. Sgsf knockout or salivary gland-specific Sgsf knockdown decrease the size of both the body and organs, phenocopying the effects of genetic ablation of salivary glands, while salivary gland-specific Sgsf overexpression increases their size. Sgsf promotes systemic growth by modulating the secretion of the insulin-like peptide Dilp2 from the brain insulin-producing cells (IPCs) and affecting mechanistic target of rapamycin (mTOR) signaling in the fat body. Altogether, our study demonstrates that Sgsf mediates the roles of salivary glands in Drosophila systemic growth, establishing an endocrine function of salivary glands.

Preparing for Transmission: Gene Regulation in Plasmodium Sporozoites

Plasmodium sporozoites are transmitted to mammals by anopheline mosquitoes and first infect the liver, where they transform into replicative exoerythrocytic forms, which subsequently release thousands of merozoites that invade erythrocytes and initiate the malaria disease. In some species, sporozoites can transform into dormant hypnozoites in the liver, which cause malaria relapses upon reactivation. Transmission from the insect vector to a mammalian host is a critical step of the parasite life cycle, and requires tightly regulated gene expression. Sporozoites are formed inside oocysts in the mosquito midgut and become fully infectious after colonization of the insect salivary glands, where they remain quiescent until transmission. Parasite maturation into infectious sporozoites is associated with reprogramming of the sporozoite transcriptome and proteome, which depends on multiple layers of transcriptional and post-transcriptional regulatory mechanisms. An emerging scheme is that gene expression in Plasmodium sporozoites is controlled by alternating waves of transcription activity and translational repression, which shape the parasite RNA and protein repertoires for successful transition from the mosquito vector to the mammalian host.

Transcriptome Analysis Reveals Functional Diversity in Salivary Glands of Plant Virus Vector, Graminella nigrifrons.

Insect salivary glands play an important role for host feeding, specifically by secreting salivary proteins for digestion and potentially modulating host defenses. Compared to other hemipterans, the significance of salivary glands is less studied in the black-faced leafhopper, Graminella nigrifrons, a crop pest that vectors several agronomically important plant viruses. To identify functionally important genes in the salivary glands of the black-faced leafhopper, we compared transcriptomes between adult salivary glands (SG) and the remaining carcasses. We identified 14,297 salivary gland-enriched transcripts and 195 predicted secretory peptides (i.e., with a signal peptide and extracellular localization characteristics). Overall, the SG transcriptome included functions such as ‘oxidoreduction’, ‘membrane transport’, and ‘ATP-binding’, which might be important for the fundamental physiology of this tissue. We further evaluated transcripts with potential contributions in host feeding using RT-qPCR. Two SG-enriched transcripts (log2 fold change > 5), GnP19 and GnE63 (a putative calcium binding protein), were significantly upregulated in maize-fed adults relative to starved adults, validating their importance in feeding. The SG-enriched transcripts of the black-faced leafhopper could play a potential role for interacting with maize and could be targets of interest for further functional studies and improve pest control and disease transmission.

Rice virus release from the planthopper salivary gland is independent of plant tissue recognition by the stylet.

The transmission of plant viruses by arthropod vectors is closely related to feeding behavior. For persistently transmitted viruses, virus release means that virus moves through the salivary gland microvillus barriers of insects into plant via the stylet. However, whether virus release is dependent on plant tissue and component recognition by the stylet is unclear. In this study, the small brown planthopper (SBPH) and two rice viruses transmitted by it were used as a model to explore this question. After the viruliferous insects penetrated a stretched membrane without plant tissue structure and ingested liquid food (rice sap, nutrient solution or water), both viruses were detected in the liquid food after only a 6 min inoculation access period, suggesting that the viruses were released from SBPH salivary gland independent of plant tissue and component recognition by the stylet. In subsequent electrical penetration graph (EPG) analysis, N4a-like and N4b-like waveforms, similar to N4a (phloem salivation before ingestion) and N4b (sieve element ingestion), were observed during SBPH penetrating the membrane, exhibiting normal feeding activity of planthopper on membrane, which further demonstrated that virus release from salivary gland was along with feeding activity, without the stylet sensing plant tissue. EPG analysis and identification of salivary proteins indicated more active feeding behavior and efficient salivation in viruliferous planthoppers. These results suggest that the rice virus is released from insect salivary gland independent of plant tissue and component recognition by the stylet, and the simple virus release mode facilitates virus transmission by vectors. © 2020 Society of Chemical Industry.

ADP ribosylation factor 1 facilitates spread of wheat dwarf virus in its insect vector.

Many plant viruses are vectored by insects in a persistent circulative manner. The insect gut and salivary gland are important barriers limiting virus spread, but the mechanisms by which viruses are able to cross the gut escape barriers of the insect remain largely unknown. Wheat dwarf virus (WDV), transmitted by Psammotettix alienus in a persistent, circulative, and nonpropagative manner, causes the most economically important virus disease in wheat. In this study, ADP ribosylation factor 1 (ARF1) was found to interact with the coat protein of WDV in a yeast two-hybrid, pull-down assay and to colocalise with virions in the gut and salivary glands of P.alienus. When transcription of ARF1 was suppressed by RNA interference, the WDV titre decreased in the haemolymph and salivary glands, and transmission efficiency decreased, but titre in the gut did not differ from that of the control. These data suggest that ARF1 of P.alienus binds to the WDV virion and helps virus spread from gut to haemolymph. Our study provides direct experimental evidence that WDV can use the existing membrane trafficking mechanism to aid its spread within the insect vector. This first analysis of the molecular interaction between WDV and its vector P.alienus contributes to understanding the mechanisms involved in circulative transmission of the virus by the leafhopper vector.

Transient Expression of Whitefly Effectors in Nicotiana benthamiana Leaves Activates Systemic Immunity Against the Leaf Pathogen Pseudomonas syringae and Soil-Borne Pathogen Ralstonia solanacearum

Infestation of plants with the phloem-feeding whitefly Bemisia tabaci modulates root microbiota and both local and systemic immunity against microbial pathogens. Specifically, aboveground whitefly infestation suppresses pathogen propagation and symptom development caused by the soil-borne pathogens Agrobacterium tumefaciens and Ralstonia solanacearum in the root system through systemic signal transduction. Therefore, we hypothesized that secreted protein(s)/non-protein factors from whitefly saliva (referred to as candidate effectors) might function as insect determinants that activate systemic acquired resistance (SAR) in the host plant. Here, we intensively screened a cDNA library constructed from mRNA from whitefly feeding on Nicotiana benthamiana leaves and selected three candidate effectors, 2G4, 2G5, and 6A10, that appear to reduce disease development caused by the aboveground pathogen Pseudomonas syringae pv. tabaci and the soil-borne pathogen R. solanacearum. Transient expression of the three candidate effector cDNAs in leaves primed the expression of SAR marker genes NbPR1a and NbPR2 in local and systemic leaves against P. syringae pv. tabaci, while leaf infiltration with 2G4 and 6A10 cDNA elicited strong defense priming of SAR markers following drench application of R. solanacearum on plant roots. In silico and qRT-PCR analyses revealed the presence of 2G5 and 6A10 transcripts in insect salivary glands. This is the first report of whitefly effectors that prime SAR against aboveground and belowground bacterial pathogens.

High-resolution analysis of multi-copy variant surface glycoprotein gene expression sites in African trypanosomes.

BackgroundAfrican trypanosomes cause lethal diseases in humans and animals and escape host immune attack by switching the expression of Variant Surface Glycoprotein (VSG) genes. The expressed VSGs are located at the ends of telomeric, polycistronic transcription units known as VSG expression sites (VSG-ESs). Each cell has many VSG-ESs but only one is transcribed in bloodstream-form parasites and all of them are inactive upon transmission to the insect vector mid-gut; a subset of monocistronic metacyclic VSG-ESs are then activated in the insect salivary gland. Deep-sequence analyses have been informative but assigning sequences to individual VSG-ESs has been challenging because they each contain closely related expression-site associated genes, or ESAGs, thought to contribute to virulence.ResultsWe utilised ART, an in silico short read simulator to demonstrate the feasibility of accurately aligning reads to VSG-ESs. Then, using high-resolution transcriptomes from isogenic bloodstream and insect-stage Lister 427 Trypanosoma brucei, we uncover increased abundance in the insect mid-gut stage of mRNAs from metacyclic VSG-ESs and of mRNAs from the unusual ESAG, ESAG10. Further, we show that the silencing associated with allelic exclusion involves repression focussed at the ends of the VSG-ESs. We also use the approach to report relative fitness costs following ESAG RNAi from a genome-scale screen.ConclusionsBy assigning sequences to individual VSG-ESs we provide new insights into VSG-ES transcription control, allelic exclusion and impacts on fitness. Thus, deeper insights into the expression and function of regulated multi-gene families are more accessible than previously anticipated.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3154-8) contains supplementary material, which is available to authorized users.

A cecropin-like antimicrobial peptide with anti-inflammatory activity from the black fly salivary glands

BackgroundSeveral antimicrobial peptides (AMPs) belonging to the cecropin family have been identified from the salivary glands of different black fly species, however, the immunological functions for these molecules were poorly understood.MethodsA novel cecropin-like antimicrobial peptide (SibaCec) was purified using reverse phase high-performance liquid chromatography (RP-HPLC) from the salivary glands of the black fly Simulium bannaense. The amino acid sequence of SibaCec was determined by a combination method of automated Edman degradation and cDNA sequencing. The morphologic changes of Gram-negative bacteria Escherichia coli treated with SibaCec were assessed by scanning electron microscopy (SEM). Quantitative PCR (qPCR) was performed to analyze the mRNA expression of the inducible NO synthase (iNOS) and pro-inflammatory cytokines. Nitric oxide (NO) generation was examined using a Griess assay and the secretion of pro-inflammatory cytokines was determined by an enzyme-linked immunosorbent assay (ELISA). The activation of extracellular signal-regulated kinase (ERK), p38, and the nuclear translocation of nuclear factor-kappaB (NF-κB) were assessed by Western blotting analysis. Circular dichroism (CD) spectroscopy was performed to evaluate the secondary structure of SibaCec in solvent environment. Interaction of SibaCec with lipopolysaccharide (LPS) was studied using fluorescein isothiocyanate (FITC)- conjugated LPS aggregates. Neutralization of LPS by SibaCec was assayed with the chromogenic limulus amebocyte lysate (LAL) test. qPCR was also used to analyze the expression of SibaCec mRNA in the salivary glands of insects after oral infection with the bacteria E.coli.ResultsSibaCec possessed potent antimicrobial activity against Gram-negative bacteria, and showed low cytotoxicity toward mammalian cells. SEM analysis indicated that SibaCec killed bacteria through the disruption of cell membrane integrity. Furthermore, SibaCec significantly inhibited lipopolysaccharide (LPS)-induced production of NO and pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interferon-1β (IL-1β) and interferon-6 (IL-6) by blocking the activation of MAPKs and NF-κB signaling pathways. It mainly adopted an α-helix conformation in membrane-mimetic environments. SibaCec could interact and neutralize LPS. Infection of black flies with bacteria caused an upregulation of the expression of SibaCec.ConclusionsThese results demonstrated that in addition to the bactericidal capacity, SibaCec can function as immune regulator, inhibiting host secretion of inflammatory factors.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1176-8) contains supplementary material, which is available to authorized users.

Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells.

In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.

Asymmetrasca decedens (Cicadellidae, Typhlocybinae), a natural vector of ‘Candidatus Phytoplasma phoenicium’

Abstract‘Candidatus Phytoplasma phoenicium’ is associated with a lethal disease of almond, peach and nectarine named almond witches'‐broom disease (AlmWB). The disease spread rapidly in Lebanon from coastal areas to elevations exceeding 1200 m, killing over 150,000 trees in a span of two decades. The mode of spread suggested the involvement of efficient vector(s) and Asymmetrasca decedens (Hemiptera, Cicadellidae) was suspected as it is the most abundant leafhopper species present in Lebanese stone fruit orchards. Living A. decedens specimens were collected from fields heavily infested by AlmWB and used in transmission trials on healthy peach almond hybrid GF‐677 and peach GF‐305 seedlings with an inoculation‐access period of 30 days. PCR analysis supported by sequencing showed that A. decedens is a carrier of the phytoplasma, and that the phytoplasma was detected in insect salivary glands and in some inoculated GF‐677 and GF‐305 seedlings. One year post‐inoculation, ‘Ca. P. phoenicium’ was detected in newly emergent leaves of inoculated seedlings. However, the characteristic symptoms of witches'‐broom were not observed. PCR amplified fragments from phytoplasma‐positive seedlings and from A. decedens samples showed 99.9% nucleotide identity in their 16S RNA region and phylogenetic analysis using a neighbour jointing tree confirmed that the phytoplasmas detected in both insects and inoculated seedlings belonged to 16SrIX‐B (D). The present manuscript is the first known report for a leafhopper vector of ‘Ca. P. phoenicium’ and shows that the incubation period of the disease in plants may be longer than 1 year. The importance of phytosanitary control measures, the adoption of a national strategy and regional cooperation in order to contain the further spread of the disease are discussed.

Latent period and transmission of "Candidatus Liberibacter solanacearum" by the potato psyllid Bactericera cockerelli (Hemiptera: Triozidae).

“Candidatus Liberibacter solanacearum” (Lso) is an economically important pathogen of solanaceous crops and the putative causal agent of zebra chip disease of potato (Solanum tuberosum L.). This pathogen is transmitted to solanaceous species by the potato psyllid, Bactericera cockerelli (Šulc), but many aspects of the acquisition and transmission processes have yet to be elucidated. The present study was conducted to assess the interacting effects of acquisition access period, incubation period, and host plant on Lso titer in psyllids, the movement of Lso from the alimentary canal to the salivary glands of the insect, and the ability of psyllids to transmit Lso to non-infected host plants. Following initial pathogen acquisition, the probability of Lso presence in the alimentary canal remained constant from 0 to 3 weeks, but the probability of Lso being present in the salivary glands increased with increasing incubation period. Lso copy numbers in psyllids peaked two weeks after the initial pathogen acquisition and psyllids were capable of transmitting Lso to non-infected host plants only after a two-week incubation period. Psyllid infectivity was associated with colonization of insect salivary glands by Lso and with Lso copy numbers >10,000 per psyllid. Results of our study indicate that Lso requires a two-week latent period in potato psyllids and suggest that acquisition and transmission of Lso by psyllids follows a pattern consistent with a propagative, circulative, and persistent mode of transmission.

Meiosis and Haploid Gametes in the Pathogen Trypanosoma brucei

SummaryIn eukaryote pathogens, sex is an important driving force in spreading genes for drug resistance, pathogenicity, and virulence [1]. For the parasitic trypanosomes that cause African sleeping sickness, mating occurs during transmission by the tsetse vector [2, 3] and involves meiosis [4], but haploid gametes have not yet been identified. Here, we show that meiosis is a normal part of development in the insect salivary glands for all subspecies of Trypanosoma brucei, including the human pathogens. By observing insect-derived trypanosomes during the window of peak expression of meiosis-specific genes, we identified promastigote-like (PL) cells that interacted with each other via their flagella and underwent fusion, as visualized by the mixing of cytoplasmic red and green fluorescent proteins. PL cells had a short, wide body, a very long anterior flagellum, and either one or two kinetoplasts, but only the anterior kinetoplast was associated with the flagellum. Measurement of nuclear DNA contents showed that PL cells were haploid relative to diploid metacyclics. Trypanosomes are among the earliest diverging eukaryotes, and our results support the hypothesis that meiosis and sexual reproduction are ubiquitous in eukaryotes and likely to have been early innovations [5].

Role of adipokinetic hormone in stimulation of salivary gland activities: The fire bug Pyrrhocoris apterus L. (Heteroptera) as a model species

The effect of adipokinetic hormone (Pyrap-AKH) in stimulating the function of insect salivary glands (SGs) in extra-oral digestive processes was studied in the firebug, Pyrrhocoris apterus L. (Heteroptera). The analyses were performed on samples of SGs and extracts of linden seeds, a natural source of the bug’s food. The SGs from 3-day old P. apterus females (when the food ingestion culminates), primarily contained polygalacturonase (PG) enzyme activity, whereas the level of lipase, peptidase, amylase and α-glucosidase was negligible. The transcription of PG mRNA and enzymatic activity were significantly increased in SGs after Pyrap-AKH treatment. The piercing and sucking of linden seeds by the bugs stimulated the intrinsic enzymatic cocktail of seeds (lipase, peptidase, amylase, glucosidase), and moreover the activity of these enzymes was significantly enhanced when the seeds were fed on by the Pyrap-AKH treated bugs. Similarly, a significant increase in PG activity was recorded in linden seeds fed on by hormonally-treated bugs or when injected by SG extract from hormonally treated ones as compared to untreated controls. The mechanism of AKH action in SGs is unknown, but likely involves cAMP (and excludes cGMP) as a second messenger, since the content of this compound doubled in SGs after Pyrap-AKH treatment. This new and as yet undescribed function of AKH in SGs is compared with the effect of this hormone on digestive processes in the midgut elucidated earlier.