Patricia V. Pietrantonio

cDNA cloning and transcriptional regulation of the vitellogenin receptor from the imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae)

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.

Cloning and expression analysis of a 5HT7-like serotonin receptor cDNA from mosquito Aedes aegypti female excretory and respiratory systems

In the mosquito Aedes aegypti, 5‐HT changes the endogenous rhythm of contractions in the female hindgut and increases fluid secretion in the larval Malpighian tubule. The role of 5‐HT as a diuretic hormone in adults has been questioned. We cloned a cDNA encoding a serotonin receptor from a female A. aegypti Malpighian tubule library that is similar to the 5‐HT7 receptor from Drosophila melanogaster. The transcript was localized in the tracheolar cells associated with the female Malpighian tubules but no signal was detectable in the tubule epithelium. Immunohistochemistry with specific antibodies confirmed the receptor expression in tracheolar cells and hindgut, and western blots of these tissues showed the expected 50 kDa band. The results suggest a role for serotonin in respiration and that this receptor may coordinate the tubule‐hindgut response to serotonin during diuresis.

The mosquito Aedes aegypti (L.) leucokinin receptor is a multiligand receptor for the three Aedes kinins.

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 ≥ 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.

Cloning and transcriptional expression of a leucokinin‐like peptide receptor from the Southern cattle tick, Boophilus microplus (Acari: Ixodidae)

Leucokinins are invertebrate neuropeptides that exhibit myotropic and diuretic activity. Only one leucokinin‐like peptide receptor is known, the lymnokinin receptor from the mollusc Lymnaeastagnalis. A cDNA encoding a leucokinin‐like peptide receptor was cloned from the Southern cattle tick, Boophilus microplus, a pest of cattle world‐wide. This is the first neuropeptide receptor known from the Acari and the second known in the subfamily of leucokinin‐like peptide G‐protein‐coupled receptors. The deduced amino acid sequence exhibits 40% identity to the lymnokinin receptor. The receptor transcript is present in all tick life stages as determined by semiquantitative reverse transcription polymerase chain reaction. We also propose that the sequence AAF50775.1 from the Drosophila melanogaster genome (CG10626) encodes the first identified insect leucokinin receptor.

Cloning of an aquaporin-like cDNA and in situ hybridization in adults of the mosquito Aedes aegypti (Diptera: Culicidae).

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.

Functional analysis of a G protein‐coupled receptor from the Southern cattle tick Boophilus microplus (Acari: Ixodidae) identifies it as the first arthropod myokinin receptor

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.

Oocyte membrane localization of vitellogenin receptor coincides with queen flying age, and receptor silencing by RNAi disrupts egg formation in fire ant virgin queens

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.

Molecular cloning and functional expression of a serotonin receptor from the Southern cattle tick, Boophilus microplus (Acari: Ixodidae)

A full‐length cDNA encoding a 5‐hydroxytryptamine (5‐HT) receptor from the Southern cattle tick, Boophilus microplus, was isolated using a strategy based on sequence homology among G protein‐coupled receptors. The deduced amino acid sequence revealed highest identity with Drosophila melanogaster 5HT‐dro2A (Z11489, 50.8%) and 5HT‐dro2B (Z11490, 49.5%) receptors. The receptor was transiently expressed in mammalian HEK293 cells, and Western blot analysis showed the expected 43.3 kDa band. In these cells, application of 5‐HT (10 µm) inhibited forskolin‐induced cAMP synthesis by 26%. The results indicate that the tick receptor is an invertebrate 5‐HT1‐like receptor that couples to Gαi protein.

Insect insulin receptors: insights from sequence and caste expression analyses of two cloned hymenopteran insulin receptor cDNAs from the fire ant

The insulin and insulin‐like growth factor (IGF) signalling (IIS) pathway in the honey bee (Apis mellifera) is linked to reproductive division of labour and foraging behaviour. Two insulin receptor genes are present in the released genomes of other social hymenopterans. Limited information is available on the IIS pathway role in ants. The predicted insulin receptor sequences from the recently released draft genome of the fire ant Solenopsis invicta (Hymenoptera: Formicidae) are incomplete and biologically significant data are also lacking. To elucidate the role of the IIS pathway in the fire ant, two putative insulin receptors (SiInR‐1 and SiInR‐2) were cloned; the first InR cDNAs cloned from social insects. Analyses of putative post‐translational modification sites in SiInRs revealed the potential for differential regulation. We investigated the transcriptional expression of both receptors at different developmental stages, castes and queen tissues. In last instar larvae and pharate pupae of workers and reproductive, transcriptional abundance of both receptors was negatively correlated with body size and nutritional status. The expression level of both receptors in different queen tissues appears to correlate with requirements for queen reproductive physiology and behaviours. This study contributes new information to the understanding of social insects because in fire ants juvenile hormone acts as a gonadotropin and workers are fully sterile, contrary to honey bees.

In vitro expression and pharmacology of the 5‐HT7‐like receptor present in the mosquito Aedes aegypti tracheolar cells and hindgut‐associated nerves

We have previously reported the cloning of a 5‐hydroxytryptamine receptor (Aedes 5‐HT7‐like receptor) from adult Aedes aegypti. For functional expression of the Aedes 5‐HT7‐like receptor, CHO‐K1 cells were stably transfected with a receptor expression construct, pC5‐HT7. The Aedes 5‐HT7‐like receptor positively coupled to Gs protein, increasing intracellular cAMP in response to 5‐HT; adenylyl cyclase activity was induced in a concentration‐dependent, saturable manner. Only 5‐HT, and not octopamine, dopamine or tyramine, caused the induction of cAMP. At 10 nm 5‐HT a weak synergism was observed between octopamine and 5‐HT. Other known agonists of the mammalian 5‐HT7 receptor were tested. Their order of potency was: 5‐HT >> 5‐CT = 8‐OH‐DPAT >> pimozide. This is the first report on the functional expression of a mosquito neurohormone receptor.