Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick–host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host ‘questing', prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.
We describe the cloning of the first hymenopteran vitellogenin receptor (VgR) cDNA from the imported fire ant, Solenopsis invicta, an invasive pest. Using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends, fragments encompassing the entire coding region of a putative VgR were cloned and sequenced. The complete 5764 bp cDNA encodes a 1782 residue protein with a predicted molecular mass of 201.3 kDa (=SiVgR). Northern blot analysis demonstrated that the 7.4 kb SiVgR transcript was present only in ovaries of reproductive females (virgin alates and queens). The temporal profile of transcriptional expression showed that SiVgR mRNA increased with age in virgin alate females and that this was up-regulated by methoprene, a juvenile hormone (JH) analogue. This suggests that the SiVgR gene is JH regulated.
A cDNA encoding a putative water channel protein, aquaporin, was cloned from a cDNA library of Aedes aegypti Malpighian tubules. The cDNA encodes a 26.11 kDa protein similar to insect aquaporins from Haematobia irritans exigua (Diptera) and Cicadella viridis (Homoptera), and to mammalian aquaporin 4. Localization of the messenger RNA (mRNA) was performed by in situ hybridization of Malpighian tubules and analysed by fluorescence and confocal microscopy. The mRNA was localized in tracheolar cells associated with the Malpighian tubules. No signal was detected in the Malpighian tubule epithelium. The molecular mechanisms for water movement between tissues and tracheoles are not yet elucidated in insects. Our results suggest a model to explain fluid movements in tracheoles during insect respiration.
In invertebrates, neuropeptide F (NPF) peptides share structural similarity with vertebrate neuropeptide Y, which regulates food consumption, circadian rhythms, anxiety, and other physiological processes. The insect neuropeptide F receptors belong to the G protein-coupled receptor (GPCR) rhodopsin family. We have cloned the fire ant putative short NPF receptor using PCR and RACE methods. The complete 2,185-bp cDNA encodes a 387-residue protein with a predicted GPCR seven transmembrane region structure. We propose that the sequence of the honey bee short NPF receptor, which has not yet been annotated, encodes a protein of 393 residues. In fire ant mated queens, receptor transcripts were detected in the brain, midgut, hindgut, Malpighian tubules, fat body, and ovaries. The highest transcriptional expression was found in the brain. The downregulation of the fire ant short NPF receptor transcriptional expression in the brain with starvation suggests that the short NPF signal transduction cascade may play a role in feeding regulation in fire ant mated queens.
A cDNA cloned from Aedes aegypti (L.) (Aedae) female Malpighian tubule (AY596453) encodes a 584 amino acid residue protein (65.2 kDa) predicted as a G protein-coupled receptor and orthologue of the drosokinin receptor from Drosophila melanogaster and highly similar to the tick Boophilus microplus myokinin receptor (AF228521). Based on the similarity to this Aedes sequence, we also propose a correction for the Anopheles gambiae protein sequence EAA05450. When expressed in CHO-K1 cells, the Aedes receptor behaved as a multiligand receptor and functionally responded to concentrations > or = 1 nM of Aedae kinins 1-3, respectively, as determined by a calcium bioluminescence plate assay and single cell intracellular calcium measurements by confocal fluorescence cytometry. Estimates of EC50 values by the plate assay were 16.04 nM for Aedae-K-3, 26.6 nM for Aedae-K-2 and 48.8 nM for Aedae-K-1 and were statistically significantly different. These results suggest that the observed differences in physiological responses to the three Aedes kinins in the Aedes isolated Malpighian tubule reported elsewhere could now be explained by differences in intracellular signalling events triggered by the different peptides on the same receptor and not necessarily due to the existence of various receptors for the three Aedes kinins.
The myokinins are invertebrate neuropeptides with myotropic and diuretic activity. The lymnokinin receptor from the snail Lymnaea stagnalis (Mollusca) has been the only previously identified myokinin receptor. We had cloned a G protein-coupled receptor (AF228521) from the tick Boophilus microplus (Arthropoda: Acari), 40% identical to the lymnokinin receptor, that we have now expressed in CHO-K1 cells. Myokinins at nanomolar concentrations induced intracellular calcium release, as measured by fluorescent cytometry and the receptor coupled to a pertussis toxin-insensitive G protein. Absence of extracellular calcium did not inhibit the fluorescence response, indicating that intracellular stores were sufficient for the initial response. Control cells only transfected with vector did not respond. We conclude that the tick receptor is the first myokinin receptor to be cloned from an arthropod.
In ant species in which mating flights are a strategic life‐history trait for dispersal and reproduction, maturation of virgin queens occurs. However, the specific molecular mechanisms that mark this transition and the effectors that control premating ovarian growth are unknown. The vitellogenin receptor (VgR) is responsible for vitellogenin uptake during egg formation in insects. In the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), virgin queens have more abundant VgR transcripts than newly mated queens, but limited egg formation. To elucidate whether the transition to egg production involved changes in VgR expression, we investigated both virgin and mated queens. In both queens, western blot analysis showed an ovary‐specific VgR band (∼ 202 kDa), and immunofluorescence analysis of ovaries detected differential VgR localization in early‐ and late‐stage oocytes. However, the VgR signal was much lower in virgin queens ready to fly than in mated queens 8 h post mating flight. In virgin queens, the receptor signal was first observed at the oocyte membrane beginning at day 12 post emergence, coinciding with the 2 weeks of maturation required before a mating flight. Thus, the membrane localization of VgR appears to be a potential marker for queen mating readiness. Silencing of the receptor in virgin queens through RNA interference abolished egg formation, demonstrating that VgR is involved in fire ant ovary development pre mating. To our knowledge, this is the first report of RNA interference in any ant species and the first report of silencing of a hymenopteran VgR.
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