Ben Warren

Sex Recognition through Midflight Mating Duets in Culex Mosquitoes Is Mediated by Acoustic Distortion

Sexual recognition through wing-beat frequency matching was first demonstrated in Toxorhynchites brevipalpis, where wing-beat frequencies of males and females are similar. Here we show frequency matching in Culex quinquefasciatus, where the wing-beat frequencies of males and females differ considerably. The wing-beat frequencies converge not on the fundamental but on the nearest shared harmonic (usually females third and males second). Frequencies in this range are, however, too high to elicit phasic sensory-neural responses from the Johnstons organ (JO) or to drive the mosquitos motor neurons. Potential cues for frequency matching are difference tones produced by nonlinear mixing of male and female flight tones in the vibrations of the mosquitos antennae. Receptor potentials and neural-motor activity were recorded in response to difference tones produced when a mosquito was stimulated simultaneously by two tones at frequencies outside the phasic response range of the JO but within range of the antennal vibrations. We demonstrate sexual recognition through matching of flight-tone harmonics in Culex mosquitoes and suggest that difference tones are used as an error signal for frequency matching beyond the frequency range of the JOs sensory-neural range. This is the first report of acoustic distortion being exploited as a sensory cue, rather than existing as an epiphenomenon.

Humming in tune: sex and species recognition by mosquitoes on the wing.

Mosquitoes are more sensitive to sound than any other insect due to the remarkable properties of their antennae and Johnston’s organ at the base of each antenna. Male mosquitoes detect and locate female mosquitoes by hearing the female’s flight tone, but until recently we had no idea that females also respond to male flight tones. Our investigation of a novel mechanism of sex recognition in Toxorhynchites brevipalpis revealed that male and female mosquitoes actively respond to the flight tones of other flying mosquitoes by altering their own wing-beat frequencies. Male–female pairs converge on a shared harmonic of their respective fundamental flight tones, whereas same sex pairs diverge. Most frequency matching occurs at frequencies beyond the detection range of the Johnston’s organ but within the range of mechanical responsiveness of the antennae. We have shown that this is possible because the Johnston’s organ is tuned to, and able to detect difference tones in, the harmonics of antennal vibrations which are generated by the combined input of flight tones from both mosquitoes. Acoustic distortion in hearing organs exists usually as an interesting epiphenomenon. Mosquitoes, however, appear to use it as a sensory cue that enables male–female pairs to communicate through a signal that depends on auditory interactions between them. Frequency matching may also provide a means of species recognition. Morphologically identical but reproductively isolated molecular forms of Anopheles gambiae fly in the same mating swarms, but rarely hybridize. Extended frequency matching occurs almost exclusively between males and females of the same molecular form, suggesting that this behavior is associated with observed assortative mating.

TRP Channels in Insect Stretch Receptors as Insecticide Targets

Defining the molecular targets of insecticides is crucial for assessing their selectivity and potential impact on environment and health. Two commercial insecticides are now shown to target a transient receptor potential (TRP) ion channel complex that is unique to insect stretch receptor cells. Pymetrozine and pyrifluquinazon disturbed Drosophila coordination and hearing by acting on chordotonal stretch receptor neurons. This action required the two TRPs Nanchung (Nan) and Inactive (Iav), which co-occur exclusively within these cells. Nan and Iav together sufficed to confer cellular insecticide responses in vivo and in vitro, and the two insecticides were identified as specific agonists of Nan-Iav complexes that, by promoting cellular calcium influx, silence the stretch receptor cells. This establishes TRPs as insecticide targets and defines specific agonists of insect TRPs. It also shows that TRPs can render insecticides cell-type selective and puts forward TRP targets to reduce side effects on non-target species.

The dynein–tubulin motor powers active oscillations and amplification in the hearing organ of the mosquito

The design principles and specific proteins of the dynein–tubulin motor, which powers the flagella and cilia of eukaryotes, have been conserved throughout the evolution of life from algae to humans. Cilia and flagella can support both motile and sensory functions independently, or sometimes in parallel to each other. In this paper we show that this dual sensory–motile role of eukaryotic cilia is preserved in the most sensitive of all invertebrate hearing organs, the Johnstons organ of the mosquito. The Johnstons organ displays spontaneous oscillations, which have been identified as being a characteristic of amplification in the ears of mosquitoes and Drosophila. In the auditory organs of Drosophila and vertebrates, the molecular basis of amplification has been attributed to the gating and adaptation of the mechanoelectrical transducer channels themselves. On the basis of their temperature-dependence and sensitivity to colchicine, we attribute the molecular basis of spontaneous oscillations by the Johnstons organ of the mosquito Culex quinquefasciatus, to the dynein–tubulin motor of the ciliated sensillae. If, as has been claimed for insect and vertebrate hearing organs, spontaneous oscillations epitomize amplification, then in the mosquito ear, this process is independent of mechanotransduction.

Rapid and Slow Chemical Synaptic Interactions of Cholinergic Projection Neurons and GABAergic Local Interneurons in the Insect Antennal Lobe

The antennal lobe (AL) of insects constitutes the first synaptic relay and processing center of olfactory information, received from olfactory sensory neurons located on the antennae. Complex synaptic connectivity between olfactory neurons of the AL ultimately determines the spatial and temporal tuning profile of (output) projection neurons to odors. Here we used paired whole-cell patch-clamp recordings in the cockroach Periplaneta americana to characterize synaptic interactions between cholinergic uniglomerular projection neurons (uPNs) and GABAergic local interneurons (LNs), both of which are key components of the insect olfactory system. We found rapid, strong excitatory synaptic connections between uPNs and LNs. This rapid excitatory transmission was blocked by the nicotinic acetylcholine receptor blocker mecamylamine. IPSPs, elicited by synaptic input from a presynaptic LN, were recorded in both uPNs and LNs. IPSPs were composed of both slow, sustained components and fast, transient components which were coincident with presynaptic action potentials. The fast IPSPs were blocked by the GABAA receptor chloride channel blocker picrotoxin, whereas the slow sustained IPSPs were blocked by the GABAB receptor blocker CGP-54626. This is the first study to directly show the predicted dual fast- and slow-inhibitory action of LNs, which was predicted to be key in shaping complex odor responses in the AL of insects. We also provide the first direct characterization of rapid postsynaptic potentials coincident with presynaptic spikes between olfactory processing neurons in the AL.

Auditory Efferent System Modulates Mosquito Hearing

The performance of vertebrate ears is controlled by auditory efferents that originate in the brain and innervate the ear, synapsing onto hair cell somata and auditory afferent fibers [1-3]. Efferent activity can provide protection from noise and facilitate the detection and discrimination of sound by modulating mechanical amplification by hair cells and transmitter release as well as auditory afferent action potential firing [1-3]. Insect auditory organs are thought to lack efferent control [4-7], but when we inspected mosquito ears, we obtained evidence for its existence. Antibodies against synaptic proteins recognized rows of bouton-like puncta running along the dendrites and axons of mosquito auditory sensory neurons. Electron microscopy identified synaptic and non-synaptic sites of vesicle release, and some of the innervating fibers co-labeled with somata in the CNS. Octopamine, GABA, and serotonin were identified as efferent neurotransmitters or neuromodulators that affect auditory frequency tuning, mechanical amplification, and sound-evoked potentials. Mosquito brains thus modulate mosquito ears, extending the use of auditory efferent systems from vertebrates to invertebrates and adding new levels of complexity to mosquito sound detection and communication.

Properties and physiological function of Ca2+-dependent K+ currents in uniglomerular olfactory projection neurons

Ca(2+)-activated potassium currents [IK(Ca)] are an important link between the intracellular signaling system and the membrane potential, which shapes intrinsic electrophysiological properties. To better understand the ionic mechanisms that mediate intrinsic firing properties of olfactory uniglomerular projection neurons (uPNs), we used whole cell patch-clamp recordings in an intact adult brain preparation of the male cockroach Periplaneta americana to analyze IK(Ca) In the insect brain, uPNs form the principal pathway from the antennal lobe to the protocerebrum, where centers for multimodal sensory processing and learning are located. In uPNs the activation of IK(Ca) was clearly voltage and Ca(2+) dependent. Thus under physiological conditions IK(Ca) is strongly dependent on Ca(2+) influx kinetics and on the membrane potential. The biophysical characterization suggests that IK(Ca) is generated by big-conductance (BK) channels. A small-conductance (SK) channel-generated current could not be detected. IK(Ca) was sensitive to charybdotoxin (CTX) and iberiotoxin (IbTX) but not to apamin. The functional role of IK(Ca) was analyzed in occlusion experiments under current clamp, in which portions of IK(Ca) were blocked by CTX or IbTX. Blockade of IK(Ca) showed that IK(Ca) contributes significantly to intrinsic electrophysiological properties such as the action potential waveform and membrane excitability.

Mosquitoes on the Wing “Tune In” to Acoustic Distortion

Our current understanding of the mating game for many mosquito species is that males aggregate in noisy mating swarms and listen with their Johnstons organs (JOs) for the deeper flight tones of approaching females, to which they are attracted. As has been demonstrated, at least for the most intensely studied vector species, the mechanical resonance of the flagellum and the frequency range of the females JO is far below that of the males flight tones. Therefore, it has been assumed that females do not use hearing to detect the presence of males. Here we reveal that this may not be the case, and that the JOs of female Culex quinquefasciatus are exquisitely tuned to low frequency distortion products in the vibrations of the antenna due to a nonlinear interaction between her own flight tones and those of a nearby male. She can hear male flight tones by virtue of, and not despite, hearing her own flight tones.