Marten J. Edwards

Differential Expression of Ixodes ricinus Tick Genes Induced by Blood Feeding or Borrelia burgdorferi Infection

Abstract Ixodes ricinus L. is the principal European vector of Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis. Subtractive hybridization was used to isolate tick genes that were induced in whole ticks after blood meals on uninfected and B. burgdorferi-infected guinea pigs. Novel cDNA clones with similarity to cytochrome c oxidase, salivary secreted protein, actin, and a cysteine protease propeptide were induced after a blood meal. Novel cDNA clones with similarity to thioredoxin peroxidases, dolichyl-phosphate β-glucosyltransferase, glutathione S-transferase, defensin, ML domain-containing protein, and von Willebrand factor were induced after B. burgdorferi infection. Virtual Northern analysis was used to verify that these genes were differentially expressed in ticks after a pathogen-infected blood meal and to detect their tissues of expression. The characterization of genes that are induced after an infected blood meal is essential for gaining an understanding of the molecular mechanisms that underlie vector-pathogen interactions.

Rapid induction by a blood meal of a carboxypeptidase gene in the gut of the mosquito Anopheles gambiae.

A search for genes induced rapidly (< 3 h) after a blood meal in the gut of the human malaria vector Anopheles gambiae led to the identification of a carboxypeptidase gene (AgCP). We report the sequence of the 1302 nt AgCP transcribed sequence, 710 nt of upstream and 585 nt of downstream DNA. The AgCP open reading frame is 60.4% identical at the nucleotide level to a blackfly, Simulium vittatum, carboxypeptidase gene. The transcriptional start site of AgCP was determined by primer extension. Expression of AgCP mRNA is detectable in the guts of pupae and sugar-fed adult female mosquitoes and is induced (approximately 10-fold) within 3 h of a blood meal. By 24 h after a blood meal, mRNA abundance returns to a level close to that present before a blood meal. Whole-mount in situ hybridization shows that AgCP mRNA expression is restricted to most or all cells of the posterior midgut. Expression of the AgCP and trypsin genes were compared and shown to differ in two fundamental ways: (1) the peak of AgCP expression after a blood meal occurs approximately 20 h before that of trypsin; and (2) induction of the AgCP gene is independent of the composition of the ingested meal whereas trypsin induction requires the presence of protein. The potential use of the AgCP promoter for driving the expression of genes that hinder the development of parasites in the mosquito gut is discussed.

Characterization of a carboxypeptidase A gene from the mosquito, Aedes aegypti

A gut‐specific carboxypeptidase A gene (AeCPA) from the mosquito, Aedes aegypti, was cloned and characterized. The gene has an open reading frame that predicts a protein of 427 amino acids, 61% of which are identical to an Anopheles gambiae carboxypeptidase A sequence. AeCPA messenger RNA (mRNA) was not detected during larval and pupal development. In situ hybridization experiments indicated that AeCPA mRNA is expressed by posterior midgut epithelial cells. In sharp contrast to An. gambiae carboxypeptidase A gene expression, AeCPA mRNA accumulates to high levels only late (≈ 16–24 h) after ingestion of a blood meal. The temporal profile of AeCPA gene induction is similar to that of Ae. aegypti late trypsin, suggesting the existence of common regulatory elements.

Permeability and disruption of the peritrophic matrix and caecal membrane from Aedes aegypti and Anopheles gambiae mosquito larvae.

In mosquito larvae, the peritrophic matrix (PM) separates the gut contents from the intestinal epithelium. This report describes a new in vivo assay for estimating PM permeability. The assay also allows for assessment of the permeability of the caecal membrane, a structure that separates each caecum from the gut lumen. Permeability was estimated by the appearance of fluorescently-labeled dextrans (size range 4,400 to 2 million Da) within the gastric caecae of mosquito larvae. While the intact peritrophic matrix was impermeable to 2 million Da dextran particles, it was permeable to dextran particles of 148 kDa and smaller. The caecal membrane appears to have considerably smaller pores, being permeable only to dextrans of 19.5 kDa and smaller. The assay was also used to devise a treatment that disrupts the PM sufficiently to allow the passage of virus-sized particles. Dithiothreitol and to a lesser extent, chitinase were effective in disrupting the PM. Cycloheximide had a small effect; Polyoxin D, Pronase and calcofluor did not alter the permeability to 2 million Da dextran particles. Disruption of the PM is discussed in the context of infecting mosquitoes with retroviral transformation vectors.

Increased per capita herbivory in the shade: Necessity, feedback, or luxury consumption

ABSTRACT Leaf chemistry and physiology vary with light environment and are often thought to directly affect herbivory patterns. Biotic (e.g., parasitoids and predators) and abiotic (e.g., temperature, relative humidity) factors known to influence herbivory also co-vary with light environment. Irrespective of mechanism, light-based differences in herbivore damage must be the result of variable herbivore abundance, per capita effects, or both. We examined the effect of light environment on leaf defence and leaf nutritional quality in Lindera benzoin (Lauraceae) and relate this to the abundance and impact of its lepidopteran herbivore Epimecis hortaria (Lepidoptera: Geometridae). In this system we consistently observe greater natural field herbivory in shade habitats relative to high light habitats, despite similar herbivore abundances; differences in herbivory are therefore most likely attributable to different per capita impacts of herbivores across environments. Potential herbivore behaviours responsible for the observed field pattern include increased foraging per day and longer developmental periods in shade habitats. A more complete understanding of observed herbivory patterns requires incorporating variation in herbivore behaviour as influenced by abiotic or biotic factors that co-vary with the different light environments. Nomenclature: Gleason & Cronquist, 1991.

A coordinated expression of biosynthetic enzymes controls the flux of juvenile hormone precursors in the corpora allata of mosquitoes.

Juvenile hormone (JH) is a key regulator of metamorphosis and ovarian development in mosquitoes. Adult female Aedes aegypti mosquitoes show developmental and dynamically regulated changes of JH synthesis. Newly emerged females have corpora allata (CA) with low biosynthetic activity, but they produce high amounts of JH a day later; blood feeding results in a striking decrease in JH synthesis, but the CA returns to a high level of JH synthesis three days later. To understand the molecular bases of these dynamic changes we combined transcriptional studies of 11 of the 13 enzymes of the JH pathway with a functional analysis of JH synthesis. We detected up to a 1000-fold difference in the levels of mRNA in the CA among the JH biosynthetic enzymes studied. There was a coordinated expression of the 11 JH biosynthetic enzymes in female pupae and adult mosquito. Increases or decreases in transcript levels for all the enzymes resulted in increases or decreases of JH synthesis; suggesting that transcript changes are at least partially responsible for the dynamic changes of JH biosynthesis observed. JH synthesis by the CA was progressively increased in vitro by addition of exogenous precursors such as geranyl-diphosphate, farnesyl-diphosphate, farnesol, farnesal and farnesoic acid. These results suggest that the supply of these precursors and not the activity of the last 6 pathway enzymes is rate limiting in these glands. Nutrient reserves play a key role in the regulation of JH synthesis. Nutritionally deficient females had reduced transcript levels for the genes encoding JH biosynthetic enzymes and reduced JH synthesis. Our studies suggest that JH synthesis is controlled by the rate of flux of isoprenoids, which is the outcome of a complex interplay of changes in precursor pools, enzyme levels and external regulators such as nutrients and brain factors. Enzyme levels might need to surpass a minimum threshold to achieve a net flux of precursors through the biosynthetic pathway. In glands with low synthetic activity, the flux of isoprenoids might be limited by the activity of enzymes with low levels of expression.

Aedes aegypti transducing densovirus pathogenesis and expression in Aedes aegypti and Anopheles gambiae larvae

Aedes aegypti densovirus (AeDNV) is a small DNA virus that has been developed into an expression and transducing vector for mosquitoes [ Afanasiev et al. (1994) Exp Parasitol 79: 322–339; Afanasiev et al. (1999) Virology 257: 62–72; Carlson et al. (2000) Insect Transgenesis: Methods and Applications (Handler, A.M. & James, A.A., eds), pp. 139–159. CRC Press, Boca Raton]. Virions carrying a recombinant genome expressing the GFP gene were used to characterize the pathogenesis of the virus in 255 individual Aedes aegypti larvae. The anal papillae of the larvae were the primary site of infection confirming previous observations ( Afanasiev et al., 1999 ; Allen‐Muira et al. (1999) Virology 257: 54–61). GFP expression was observed in most cases to spread from the anal papillae to cells of the fat body, and subsequently to many other tissues including muscle fibers and nerves. Infected anal papillae were also observed to shrink, or melanize and subsequently fall off in a virus dependent manner. Three to four day‐old larvae were less susceptible to viral infection and, if infected, were more likely to survive into adulthood, with 14% of them still expressing GFP as adults. Higher salt concentrations of 0.10–0.15 m inhibited viral infection. Anopheles gambiae larvae also showed infection of the anal papillae (17%) but subsequent viral dissemination did not occur. The persistence of the reporter gene expression into adulthood of Aedes aegypti indicates that transduction of mosquito larvae with recombinant AeDNV may be a means of introducing a gene of interest into a mosquito population for transient expression.

A gut‐specific serine protease from the malaria vector Anopheles gambiae is downregulated after blood ingestion

A chymotrypsin‐like serine protease gene (AgChyL) was cloned from the mosquito Anopheles gambiae by a polymerase chain reaction (PCR)‐based subtractive cDNA cloning strategy. AgChyL messenger RNA (mRNA) is abundant in the adult female gut prior to, and for 8 h following, a blood meal. During the peak of digestion, from 12 to 24 h following a blood meal, AgChyL mRNA abundance decreased to barely detectable levels. AgChyL mRNA was abundant again by 48 h following a blood meal. Recombinant pro‐AgChyL was expressed in Escherichia coli. The pro‐enzyme can be activated by trypsin. Activated AgChyL cleaves the synthetic chymotrypsin substrate succinyl‐ l‐Ala‐Ala‐Pro‐Phe‐nitroanilide, but not two other synthetic chymotrypsin substrates or synthetic trypsin and elastase substrates. The potential role of AgChyL in the coordination of An. gambiae digestion is discussed.

Relatively low prevalence of Babesia microti and Anaplasma phagocytophilum in Ixodes scapularis ticks collected in the Lehigh Valley region of eastern Pennsylvania

Several human pathogens are transmitted by the blacklegged tick, Ixodes scapularis. These include the spirochetes that cause Lyme disease (Borrelia burgdorferi) which is endemic to the Lehigh Valley region of eastern Pennsylvania. Emerging and currently rare tick-borne diseases have been of increasing concern in this region, including tick-borne relapsing fever (caused by Borrelia miyamotoi), human granulocytic anaplasmosis (caused by Anaplasma phagocytophilum), and human babesiosis (caused by Babesia microti). Real-time PCR assays and in some instances, conventional PCR followed by DNA sequencing, were used to screen 423 DNA samples that were prepared from questing adult and nymph stage I. scapularis ticks for infection with four tick-borne human pathogens. B. burgdorferi was detected in 23.2% of the sampled ticks, while B. miyamotoi, B. microti and a human variant of A. phagocytophilum were detected in less than 0.5% of the ticks. Our results are consistent with those expected in a region where Lyme disease is prevalent and human cases of tick-borne relapsing fever, babesiosis and human granulocytic anaplasmosis are not currently widespread. It is expected that this study will serve as a baseline for future studies of tick-borne pathogens in an area that is in close proximity to regions of high endemicity for Lyme disease, human granulocytic anaplasmosis and human babesiosis.

The periodical cicada four-year acceleration hypothesis revisited and the polyphyletic nature of Brood V, including an updated crowd-source enhanced map (Hemiptera: Cicadidae: Magicicada)

The periodical cicadas of North America (Magicicada spp.) are well-known for their long life cycles of 13 and 17 years and their mass synchronized emergences. Although periodical cicada life cycles are relatively strict, the biogeographic patterns of periodical cicada broods, or year-classes, indicate that they must undergo some degree of life cycle switching. We present a new map of periodical cicada Brood V, which emerged in 2016, and demonstrate that it consists of at least four distinct parts that span an area in the United States stretching from Ohio to Long Island. We discuss mtDNA haplotype variation in this brood in relation to other periodical cicada broods, noting that different parts of this brood appear to have different origins. We use this information to refine a hypothesis for the formation of periodical cicada broods by 1- and 4-year life cycle jumps.