Behavioral responses of mice following scorpion venom injection.

<p>Behavioral responses of mice following scorpion venom injection.</p

Arizona bark scorpion venom resistance in the pallid bat, <i>Antrozous pallidus</i>

<div><p>The pallid bat (<i>Antrozous pallidus</i>), a gleaning bat found in the western United States and Mexico, hunts a wide variety of ground-dwelling prey, including scorpions. Anecdotal evidence suggests that the pallid bat is resistant to scorpion venom, but no systematic study has been performed. Here we show with behavioral measures and direct injection of venom that the pallid bat is resistant to venom of the Arizona bark scorpion, <i>Centruroides sculpturatus</i>. Our results show that the pallid bat is stung multiple times during a hunt without any noticeable effect on behavior. In addition, direct injection of venom at mouse LD₅₀ concentrations (1.5 mg/kg) has no effect on bat behavior. At the highest concentration tested (10 mg/kg), three out of four bats showed no effects. One of the four bats showed a transient effect suggesting that additional studies are required to identify potential regional variation in venom tolerance. Scorpion venom is a cocktail of toxins, some of which activate voltage-gated sodium ion channels, causing intense pain. Dorsal root ganglia (DRG) contain nociceptive neurons and are principal targets of scorpion venom toxins. To understand if mutations in specific ion channels contribute to venom resistance, a pallid bat DRG transcriptome was generated. As sodium channels are a major target of scorpion venom, we identified amino acid substitutions present in the pallid bat that may lead to venom resistance. Some of these substitutions are similar to corresponding amino acids in sodium channel isoforms responsible for reduced venom binding activity. The substitution found previously in the grasshopper mouse providing venom resistance to the bark scorpion is not present in the pallid bat, indicating a potentially novel mechanism for venom resistance in the bat that remains to be identified. Taken together, these results indicate that the pallid bat is resistant to venom of the bark scorpion and altered sodium ion channel function may partly underlie such resistance.</p></div

Dose and indication of response to Arizona bark scorpion venom when injected into pallid bats.

<p>Dose and indication of response to Arizona bark scorpion venom when injected into pallid bats.</p

Comparison of selected extracellular loops in Nav1.7 known to be involved in scorpion toxin binding.

<p>While Nav1.7 displays normal activity in the grasshopper mouse, it may be altered in the pallid bat providing venom resistance. (A) Schematic of Nav1.7 showing known scorpion toxin binding regions and regions of special note in the pallid bat; red are known alpha scorpion toxin binding regions and blue are known beta scorpion toxin binding regions. (B) Extracellular IS5-S6. (C) Extracellular region IIS1-S2. (D) IIS3-S4. (E) Extracellular region IVS5-S6.</p

Time required for bats to subdue scorpions or abandon attack and the number of observed stings during each encounter.

<p>Time required for bats to subdue scorpions or abandon attack and the number of observed stings during each encounter.</p

ETH evokes sequential activation of kinin and CAMB neurons.

<p>(A) Immunohistochemical staining to verify Gal4 expression in both kinin and CAMB neurons (Scale bars = 50μm). Kinin neurons (Kinin-Gal4, far left), CAMB neurons (Pburs-Gal4, left), and double Gal4 (right) were labeled by GFP using <i>pbur-Gal4</i>, <i>kinin-Gal4</i> or <i>pburs;kinin</i> combination <i>Gal4</i> and <i>UAS-GFP</i>. Far right: Schematic diagram showing relative position of CAMB neurons (AN1-4) and kinin neurons (AN 1–7). Note that kinin neurons project axons to a terminal plexus (TP, neuropil) in AN9 (arrow). Kinin neurons project axons posteriorly to TP and then turn anteriorly along the ventral midline. SN: subesophageal neuromeres; TN: thoracic neuromeres; AN: Abdominal neuromeres. (B) Ca²⁺ dynamics in kinin and CAMB neurons by ETH. (B1) Representative recordings of intracellular Ca²⁺ dynamics in kinin neurons (AN7, TP) and CAMB (AN3, 4) following exposure to ETH 1 & 2 (300 nM each) applied at time 0 (downward arrows). Following ETH application, kinin cell bodies in AN 7 and TP show robust and highly synchronized calcium oscillations after characteristic delays. CAMB neurons become active shortly after termination of kinin neuron activity. (B2) Video image shows locations of cell bodies and TP where Ca²⁺ dynamics were recorded (Top). Time-lapse video images captured during Ca²⁺ responses (bottom): timing of video image recordings (a-h) are indicated by vertical arrows in B1 (faint red). (C) Onset and termination of Ca²⁺ responses in kinin and CAMB neurons induced by ETH 1 & 2. Upon exposure to ETH1 and ETH2 (300 nM each; left), kinin and CAMB neurons are activated sequentially at 8.5 min and 20.0 min respectively. Doubling ETH concentration (600 nM each of ETH1 and ETH2, right) accelerates kinin and CAMB neuron activation, but sequential activity is maintained (6.0 min and 12.0 min respectively). Note that CAMB neuron activity lasts more than 40 min.</p

Rescheduling Behavioral Subunits of a Fixed Action Pattern by Genetic Manipulation of Peptidergic Signaling

<div><p>The ecdysis behavioral sequence in insects is a classic fixed action pattern (FAP) initiated by hormonal signaling. Ecdysis triggering hormones (ETHs) release the FAP through direct actions on the CNS. Here we present evidence implicating two groups of central ETH receptor (ETHR) neurons in scheduling the first two steps of the FAP: kinin (aka drosokinin, leucokinin) neurons regulate pre-ecdysis behavior and CAMB neurons (<b>C</b>CAP, <b>A</b>stCC, <b>M</b>IP, and <b>B</b>ursicon) initiate the switch to ecdysis behavior. Ablation of kinin neurons or altering levels of ETH receptor (ETHR) expression in these neurons modifies timing and intensity of pre-ecdysis behavior. Cell ablation or ETHR knockdown in CAMB neurons delays the switch to ecdysis, whereas overexpression of ETHR or expression of pertussis toxin in these neurons accelerates timing of the switch. Calcium dynamics in kinin neurons are temporally aligned with pre-ecdysis behavior, whereas activity of CAMB neurons coincides with the switch from pre-ecdysis to ecdysis behavior. Activation of CCAP or CAMB neurons through temperature-sensitive TRPM8 gating is sufficient to trigger ecdysis behavior. Our findings demonstrate that kinin and CAMB neurons are direct targets of ETH and play critical roles in scheduling successive behavioral steps in the ecdysis FAP. Moreover, temporal organization of the FAP is likely a function of ETH receptor density in target neurons.</p></div

Altered ETHR expression in central ensembles modifies scheduling of the ecdysis FAP.

<p>(A) Knockdown of ETHR expression using three independent ETHR-RNAi lines: <i>UAS-ETHR-IR1</i>, <i>UAS-ETHR-IR2</i>, and <i>UAS-ETHR-sym</i> carrying <i>UAS-Dicer2</i>. ETHR knockdown in kinin neurons reduced pre-ecdysis duration. ETHR knockdown in CAMB neurons (<i>Pburs-Gal4</i>) delays the switch to ecdysis behavior. A model below depicts how ETHR knockdown in kinin neurons or CAMB neurons changes pre-ecdysis duration. Bars represent mean time (± SEM, min) of the switch from pre-ecdysis to ecdysis onset relative to pre-ecdysis initiation (time zero). Data was analyzed using Mann-Whitney test (* <i>p</i> < 0.01; ** <i>p</i> < 0.001; *** <i>p</i> < 0.0001.) (B) ETHR over-expression in kinin neurons causes increased pre-ecdysis duration due to premature onset of pre-ecdysis. On the other hand, over-expression of ETHR in CAMB neurons accelerates the switch to ecdysis behavior due to increased sensitivity to ETH. See model below depicting how ETHR overexpression in kinin neurons or CAMB neurons affects pre-ecdysis duration. Error-bars represent standard error of mean (S.E.M). Data was analyzed using Mann-Whitney test (** <i>P</i> < 0.001, *** <i>P</i> < 0.0001).</p

Role of G-Protein-mediated signal transduction in timing of the switch to ecdysis behavior.

<p>(A) Inhibition of Gαo signaling using two different fly lines expressing the pertussis toxin gene (<i>PTX(2)</i>, <i>PTX(3)</i> on 2^nd and 3^rd chromosomes, respectively). (B) Enhancement of Gαo signaling by overexpression of a constitutively active form (<i>Gαo</i>^<i>Q205L</i>). (C) Overexpression of Gαq signaling in CCAP and kinin neurons by expression of a wild type Gαq. Error-bars represent standard error of mean (S.E.M). Data was analyzed using Mann-Whitney test (** <i>P</i> < 0.001, *** <i>P</i> < 0.0001).</p

Flies with impaired kinin or CAMB signaling show significant defects in the ecdysis FAP.

<p>(A) Roles for kinin in the ecdysis behavioral sequence were investigated by analysis of behavioral defects in kinin cell-killing (CK) flies and homozygous <i>piggyBac</i>-insertional kinin receptor mutant flies (<i>Lkr</i>^{<i>f02594</i>} / <i>Lkr</i>^{<i>f02594</i>}). In both instances, pre-ecdysis durations were highly variable. Precise flip-out of <i>piggyBac</i> insertion by <i>piggyBac</i> transposase rescued normal pre-ecdysis behavior. Complementation testing with a kinin-receptor-gene deficient line [<i>Df(3L)Exel6105</i>] also showed high variation in pre-ecdysis duration. The small black arrowheads represent pre-ecdysis durations of individual animals. Error-bars represent standard deviation (SD). (B) Relative expression ratio of kinin receptor genes in control and homozygous <i>Lkr</i>^{<i>f02594</i>}. Kinin receptor mutant <i>Lkr</i>^{<i>f02594</i>} showed significant reduction (26.2%) in gene expression level. Error-bars represent standard error of mean (SEM) (* <i>P</i> < 0.01; Student’s t-test). (C) EGFP staining patterns of kinin and CAMB (Pburs-Gal4) neurons. (D) Flies bearing targeted cell-killing (CK) of CAMB neurons exhibit prolonged pre-ecdysis and complete absence of ecdysis and post-ecdysis. Pre-ecdysis behavior begins with the normal frequency of rhythmic contractions, but the behavior weakens gradually after 10 min, ending at ~26 min. Error-bars represent standard deviation (SD).</p