Functional validation of the carbon dioxide receptor in labial palps of Helicoverpa armigera moths

Adult moths possess an organ in their labial palps, the labial-palp pit organ, which is specialized for sensing carbon dioxide (CO₂). They use CO₂ as a cue to detect healthy plants and find food or lay eggs on them. The molecular bases of the CO₂ receptor in Drosophila melanogaster and Aedes aegypti have been reported, but the molecular mechanisms of the CO₂ receptor in Lepidoptera remains elusive. In this study, we first re-examined three putative Helicoverpa armigera CO₂ gustatory receptor genes (HarmGr1, HarmGr2, and HarmGr3), and then analyzed expression patterns of them. RT-PCR results verified they were predominantly expressed in the labial palps of H. armigera. Thus, we used in situ hybridization to localize the expression of three genes in the labial palps. We found that all three genes were co-expressed in the same cells of the labial palps. Next, we employed the Xenopus laevis oocyte expression system and the two-electrode voltage-clamp recording to study the function of the three genes. Results showed that only oocytes co-expressing HarmGr1 and HarmGr3 or co-expressing HarmGr1, HarmGr2 and HarmGr3 gave robust responses to NaHCO₃. Finally, we confirmed that the sensory cells in labial palps of both females and males show dose dependent responses to CO₂ stimuli by using single sensillum recording. Our work uncovers that HarmGr1 and HarmGr3 are indispensable and sufficient for CO₂ sensing in labial palps of H. armigera.     

Gustatory receptor 22e is essential for sensing chloroquine and strychnine in Drosophila melanogaster

Chloroquine, an amino quinolone derivative commonly used as an anti-malarial drug, is known to impart an unpleasant taste. Little research has been done to study chloroquine taste in insects, therefore, we examined both the deterrant properties and mechanisms underlying chloroquine perception in fruit flies. We identified the antifeedant effect of chloroquine by screening 21 gustatory receptor (Grs) mutants through behavioral feeding assays and electrophysiology experiments. We discovered that two molecular sensors, GR22e and GR33a, act as chloroquine receptors, and found that chloroquine-mediated activation of GRNs occurs through S-type sensilla. At the same time, we successfully recapitulated the chloroquine receptor by expressing GR22e in ectopic gustatory receptor neurons. We also found that GR22e forms a part of the strychnine receptor. We suggest that the Drosophila strychnine receptor might have a very complex structure since five different GRs are required for strychnine-induced action potentials.     

Gustatory receptors required for sensing umbelliferone in Drosophila melanogaster

Studies of taste modality using the animal model Drosophila melanogaster have elucidated a number of uncharacterized mechanisms of sensory responses. Gustatory receptors expressed in taste organs are not only responsible for the acceptance and rejection of different foods, but are also involved in the process of selecting an oviposition site. This contact-chemosensation is essential for animals to discriminate between nutritious and contaminated foods. In this study, we characterized the function of gustatory receptors that play a dual role in feeding and oviposition using the plant metabolite umbelliferone. The combined electrophysiological and behavioral evidence demonstrated that two broadly tuned gustatory receptors, GR33a and GR66a, and one narrowly tuned gustatory receptor, GR93a, are all required to generate a functional umbelliferone receptor.     

Identification of a gustatory receptor tuned to sinigrin in the cabbage butterfly Pieris rapae

Glucosinolates are token stimuli in host selection of many crucifer specialist insects, but the underlying molecular basis for host selection in these insects remains enigmatic. Using a combination of behavioral, electrophysiological, and molecular methods, we investigate glucosinolate receptors in the cabbage butterfly Pieris rapae. Sinigrin, as a potent feeding stimulant, elicited activity in larval maxillary lateral sensilla styloconica, as well as in adult medial tarsal sensilla. Two P. rapae gustatory receptor genes PrapGr28 and PrapGr15 were identified with high expression in female tarsi, and the subsequent functional analyses showed that Xenopus oocytes only expressing PrapGr28 had specific responses to sinigrin; when ectopically expressed in Drosophila sugar sensing neurons, PrapGr28 conferred sinigrin sensitivity to these neurons. RNA interference experiments further showed that knockdown of PrapGr28 reduced the sensitivity of adult medial tarsal sensilla to sinigrin. Taken together, we conclude that PrapGr28 is a gustatory receptor tuned to sinigrin in P. rapae, which paves the way for revealing the molecular basis of the relationships between crucifer plants and their specialist insects.     

Behavioral and electrophysiological studies on the sexually biased synergism between oligosaccharides and phospholipids in gustatory perception of nuptial secretion by the German cockroach

Females of Blattella germanica feed compulsively on a nuptial secretion from the male's eighth tergal gland (TG-8) during courtship. Using TG-8 extract and its essential ingredients, maltotriose (MT) and 1,2-dioleoylphosphatidylcholine (PC), we investigated the perception of the secretion by gustatory sensilla in both sexes. Female-biased chemosensitivity was found in the feeding responses to the TG-8 extract. The TG-8 extract induced specific impulses in four functionally different receptor cells: the sugar, salt and other two types of receptor cells in a single gustatory sensillum on the paraglossae in both sexes. The impulse frequencies of the sugar receptor cell and a receptor cell of unknown type were significantly higher in females than those in males. There were no sexual differences in the behavioral and electrophysiological responses to MT alone; no responses were elicited by PC. However, a mixture of MT and PC elicited the behavioral responses more strongly than MT in females. The impulse frequency of sugar receptor cells toward the mixture of MT and PC also increased in females but not in males. These results suggest that the synergistic effect of PC on ‘sweetness’ of MT in the female cockroach contributes to her compulsive nuptial feeding. The female-biased gustatory sensitivity seems to ensure successful coupling during the nuptial feeding.     

Thermoregulatory,behavioral and productive responses of laying hens supplemented with different types and dosages of phytases raised in a hot environment: An integrative approach

This study had the following objectives: (i) to evaluate the thermoregulatory and behavioral responses of light laying hens supplemented with different types and dosages of phytases in the two day shifts; and (ii) to integrate the thermoregulatory and behavioral responses with performance of these birds raised in a hot environment. 270 light laying hens of the Hy-Line White lineage, with a body weight of 1.60 ± 0.092 kg were distributed in a completely randomized design in a 2 × 2 + 1 factorial model with two types of phytases (bacterial and fungal) and two dosages (450 and 900 FTU), and a control diet. The day shift (morning and afternoon) was considered as a fixed effect in the factorial arrangement. Principal component analysis (P_CA), correspondence analysis (C_A) and canonical discriminant analysis (C_DA) were used. There was no interaction (P > 0.05) between phytases and dosages for thermoregulatory responses. Respiratory rate (R_R), cloacal temperature (C_T), and surface temperature with feathers (S_TWF) and featherless (S_TF) were higher (P < 0.001) in the afternoon. Birds show different thermoregulatory and behavioral responses in the two shifts of the day. We also observed that birds supplemented with bacterial and fungal phytase showed similar thermoregulatory and behavioral responses to the control group in both day shifts. Expression of the “eating” activity was greater in the morning, while the birds remained sitting longer in the afternoon. Egg production was higher (P < 0.001) in birds supplemented with bacterial phytase. The phytase dosages had no effect on thermoregulatory, behavioral or performance responses. Egg production, feed conversion per dozen eggs corresponded to 81.1% of the differences between bacterial and fungal phytase supplementation and group control. Thus, we conclude that: (i) phytase dietary supplementation has no effect on the thermoregulatory responses of laying hens reared in a hot environment; (ii) birds supplemented with bacterial phytase showed higher egg production; and (iii) phytases (450 and 900 FTU) do not interfere with productive, behavioral and thermoregulatory responses.     

A Calcium-Receptor Agonist Induces Gustatory Neural Responses in Bullfrogs

The effect of calcium-sensing receptor (CaR) agonists on frog gustatory responses was studied using glossopharyngeal nerve recording and whole-cell patch-clamp recording of isolated taste disc cells. Calcimimetic NPS R-467 dissolved in normal saline solution elicited a large transient response in the nerve. The less active enantiomer of the compound, NPS S-467 induced only a small neural response. The EC₅₀ for NPS R-467 was about 20 μM. Cross-adaptation experiments were performed to examine the effect of 30 μM NPS R-467 and 100 μM quinine on the gustatory neural response. The magnitude of the R-467-induced response after adaptation to quinine was approximately equal to that after adaptation to normal saline solution, indicating that the receptor site for NPS R-467 is different from the site for quinine. NPS R-467 (100 μM) also induced an inward current accompanied with conductance increase and large depolarization in two (13%) of 15 rod cells, and a sustained decrease in outward current and small depolarization in six (40%) other rod cells. NPS S-467 (100 μM) induced a sustained decrease in outward current and depolarization in five (50%) of 10 rod cells. Another calcimimetic cinacalcet (100 μM) induced an inward current accompanied with conductance increase in three (27%) of 11 rod cells. The results suggest that NPS R-467 induces neural responses through cell responses unrelated to a resting K⁺ conductance decrease.     

The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test

Pre-clinical and clinical studies have employed treatment with glucocorticoid receptor (GR) antagonists in an attempt to limit the deleterious behavioral and physiological effects of excess glucocorticoids. Here, we examined the effects of GR antagonists on neuroendocrine and behavioral stress responses, using two compounds: mifepristone, a GR antagonist that is also a progesterone receptor antagonist, and CORT 108297, a specific GR antagonist lacking anti-progestin activity. Given its well-documented impact on neuroendocrine and behavioral stress responses, imipramine (tricyclic antidepressant) served as a positive control. Male rats were treated for five days with mifepristone (10 mg/kg), CORT 108297 (30 mg/kg and 60 mg/kg), imipramine (10 mg/kg) or vehicle and exposed to forced swim test (FST) or restraint stress. Relative to vehicle, imipramine potently suppressed adrenocorticotropin hormone (ACTH) responses to FST and restraint exposure. Imipramine also decreased immobility in the FST, consistent with antidepressant actions. Both doses of CORT 108297 potently suppressed peak corticosterone responses to FST and restraint stress. However, only the higher dose of CORT 108297 (60 mg/kg) significantly decreased immobility in the FST. In contrast, mifepristone induced protracted secretion of corticosterone in response to both stressors, and modestly decreased immobility in the FST. Taken together, the data indicate distinct effects of each compound on neuroendocrine stress responses and also highlight dissociation between corticosterone responses and immobility in the FST. Within the context of the present study, our data suggest that CORT 108297 may be an attractive alternative for mitigating neuroendocrine and behavioral states associated with excess glucocorticoid secretion.     

Targeting the intersubunit cavity of Plasmodium falciparum glutathione reductase by a novel natural inhibitor: Computational and experimental evidence

Glutathione reductase (GR), a homodimeric FAD-dependent disulfide reductase, is essential for redox homeostasis of the malaria parasite Plasmodium falciparum and has been proposed as an antimalarial drug target. In this study we performed a virtual screening against PfGR, using the structures of about 170,000 natural compounds. Analysis of the two top-scoring molecules, TTB and EPB, indicated that these ligands are likely to interact with the homodimer intersubunit cavity of PfGR with high binding energy scores of −9.67 and −9.60 kcal/mol, respectively. Both compounds had a lower affinity for human GR due to differences in structure and electrostatic properties. In order to assess the putative interactions in motion, molecular dynamics simulations were carried out for 30 ns, resulting in TTB being more dynamically and structurally favored than EPB. A closely related compound MDPI 21618 was tested on recombinant PfGR and hGR, resulting in IC₅₀ values of 11.3 ± 2.5 μM and 10.2 ± 1.7 μM, respectively. Kinetic characterization of MDPI 21618 on PfGR revealed a mixed-type inhibition with respect to glutathione disulfide (K_i = 9.7 ± 2.3 μM) and an uncompetitive inhibition with respect to NADPH. Furthermore, MDPI 21618 was found to inhibit the growth of the chloroquine-sensitive P. falciparum strain 3D7 with an IC₅₀ of 3.2 ± 1.9 μM and the chloroquine-resistant Dd2 strain with an IC₅₀ of 3.2 + 1.6 μM. In drug combination assays with chloroquine, artemisinin, or mefloquine MDPI 21618 showed an antagonistic action, which might suggest partially overlapping routes of action. This study further substantiates research on PfGR as a potential antimalarial drug target.     

3D models of lamprey corticoid receptor complexed with 11-deoxycortisol and deoxycorticosterone

The serum of Atlantic sea lamprey, a basal vertebrate, contains two corticosteroids, 11-deoxycortisol and deoxycorticosterone. Only 11-deoxycortisol has high affinity [K_d ∼ 3 nM] for the corticoid receptor [CR] in lamprey gill cytosol. To investigate the binding of 11-deoxycortisol to the CR, we constructed 3D models of lamprey CR complexed with 11-deoxycortisol and deoxycorticosterone. These 3D models reveal that Leu-220 and Met-299 in lamprey CR have contacts with the 17α-hydroxyl on 11-deoxycortisol. Lamprey CR is the ancestor of the mineralocorticoid receptor [MR] and glucocorticoid receptor [GR]. Unlike human MR and human GR, the 3D model of lamprey CR finds a van der Waals contact between Cys-227 in helix 3 and Met-264 in helix 5. Mutant human MR and GR containing a van der Waals contact between helix 3 and helix 5 display enhanced responses to progesterone and glucocorticoids, respectively. We propose that this interaction was present in the CR and lost during the evolution of the MR and GR, leading to changes in their response to progesterone and corticosteroids, respectively.     

Steroid hormones promote bovine oocyte growth and connection with granulosa cells

Many approaches have been investigated for growing oocytes in vitro in mammals. To support oocyte growth in vitro, the culture systems must meet certain conditions for maintaining connections between oocytes and surrounding granulosa cells. The aims of this study were to determine the effects of combinations of 17β-estradiol (E₂) and androstenedione (A₄) on in vitro growth of bovine oocytes and to determine the number of connections between the oocyte and granulosa cells. Oocyte–granulosa cell complexes (OGCs) collected from early antral follicles (0.4−0.7 mm in diameter) were cultured for 14 days in a medium with different concentrations of E₂ and A₄, either alone or in combinations. We then assessed the number of transzonal projections (TZPs), which extend from granulosa cells through the zona pellucida to the oolemma. During in vitro growth culture, OGC structures were maintained in the medium with steroid hormones. The mean diameter of oocytes grown in the medium with both E₂ and A₄ was increased from 95.8 μm to around 120 μm, larger than oocytes grown without steroid hormones (109.9 μm) and similar in size to in vivo fully grown oocytes (119.4 μm) from 4- to 6-mm antral follicles. In subsequent in vitro maturation culture (22 hours), 30% (12 of 40) and 34% (14 of 41) of oocytes grown with E₂ or A₄ alone, respectively, matured to metaphase II; meanwhile, oocytes grown with a combination of E₂ and A₄ matured to metaphase II at a high rate (58%, 23 of 40). Growing oocytes isolated from early antral follicles had many uniformly distributed TZPs throughout the zona pellucida. After 14 days of culture, there was a significant decrease in the number of TZPs in oocytes grown without steroid hormones, whereas the number of TZPs was maintained in oocytes grown with steroid hormones. In particular, oocytes grown with E₂ alone or with a combination of E₂ and A₄ had numbers of TZPs similar to oocytes before growth culture. In conclusion, a combination of E₂ and A₄ maintained the connections between oocytes and granulosa cells during in vitro growth culture of bovine oocytes for 14 days, resulting in the complete oocyte growth and the acquisition of meiotic competence in more than half the oocytes.     

Do hummingbirds have a sweet-tooth? Gustatory sugar thresholds and sugar selection in the broad-billed hummingbird Cynanthus latirostris

Nectar is a solution of mainly three sugars: sucrose, glucose and fructose. Studies have demonstrated that pollinators have preferences according to the sugar composition presented in their diet, and these preferences may be caused by sugar assimilation capacities. However, sugar flavor could also play an important role for sugar preferences of nectar-feeding animals. We evaluated the sugar gustatory thresholds of the broad-billed hummingbird Cynanthus latirostris for sucrose, glucose, fructose and a 1:1 mixture of glucose-fructose. We presented eight C. latirostris to paired feeders containing either a sugar solution or pure water. Additionally, we conducted sugar preference tests at three different concentrations (146, 730 and 1022 mmol L^− 1), to relate sugar preferences with sugar gustatory thresholds. C. latirostris had different gustatory thresholds for the three different sugars tested. At low sugar concentrations (146 mmol L^− 1), sugar selection followed the gustatory thresholds. Hummingbird sugar preference patterns can be affected by different mechanisms, both pre- and post-ingestive. At low concentrations gustatory thresholds may play an important role to determine sugar selection. However, at intermediate and high concentrations, sugar assimilation rates, and velocity of food processing generated by osmotic constraints, can be the mechanisms that explain the sugar selection of these animals.     

Divergent behavioral and electrophysiological taste responses in the mid-legs of adult butterflies, Vanessa indica and Argyreus hyperbius

Adult nymphalid butterflies possess sensilla trichodea (ST) that perceive taste in their walking legs. We examined whether the gustatory responses to mid-leg tarsal stimulation were different between Vanessa indica (rotting-food feeder) and Argyreus hyperbius (flower-nectar feeder). Sucrose, fructose, and glucose elicited behavioral responses (proboscis extension reflex: PER) and electrophysiological responses (spikes) from ST. Sugar responsiveness was similar in both species, where sucrose was the most stimulatory. Two fermentation products, ethanol and acetic acid, never induced PERs but elicited large-amplitude spikes at a concentration of >1% (w/v). The two species significantly differed in responsiveness to the binary mixtures of sucrose and the fermentation products. Ethanol enhanced the sugar responses of V. indica but slightly inhibited those of A. hyperbius. Although acetic acid suppressed the sugar responses of both species, V. indica was less susceptible than A. hyperbius. When concentration of the fermentation products increased, binary mixtures evoked large spikes together with small ones regarded as the sucrose responses. Unlike the proboscal sensilla in our previous study, the tarsal ST of both species unambiguously responded to fermentation products. These results demonstrate that the tarsal gustatory sense of V. indica is adaptive to the use of rotting foods.     

Tarsal taste neurons of Helicoverpa assulta (Guenée) respond to sugars and amino acids, suggesting a role in feeding and oviposition

Helicoverpa assulta and Helicoverpa armigera are sibling species with different host-plant ranges. We have previously reported electrophysiological and behavioral responses of H.armigera to sugars and amino acids. Here we describe a parallel study performed on H. assulta and compare the results obtained with the two species. In females, fourteen gustatory chemosensilla, identified on one ventrolateral side of the fifth tarsomere were stimulated with sucrose, glucose, fructose, maltose, myo-inositol, and the twenty common amino acids, using the tip-recording technique. The taste receptor neurons in eight chemosensilla were identified sensitive to the sugars, myo-inositol, Lys, Glu, Arg, Trp, and Ser which all induced proboscis extension reflex (PER) when tarsi were stimulated. There was a positive correlation between electrophysiological activities and PER responses triggered by sucrose. No stimulatory effect on oviposition was observed with sugar or amino acid mixtures. In males, three chemosensilla showed responses to the four sugars, but generally weaker than in females. The major difference of the two species was the variety of amino acids triggering electrophysiological responses. The stimulatory effect of sugars and amino acids on H.assulta was also generally weaker than that on H. armigera.     

Embryo development after vitrification of immature and in vitro-matured equine oocytes

Effects of meiotic stage and cumulus status on development of equine oocytes after vitrification was evaluated. Immature oocytes with corona radiata (IMM); in vitro-matured oocytes with corona radiata (MAT CR+); and in vitro-matured oocytes denuded of cumulus (MAT CR-) were vitrified using the Cryotech® method. Warming medium was equilibrated either in 5% CO₂ or Air. IMM oocytes underwent in vitro maturation after warming. Recovery, survival, and maturation rates, and cleavage and blastocyst rates after ICSI, were evaluated. Recovery was higher for oocytes warmed in CO₂- than Air-equilibrated medium (86 ± 3 vs. 76.9 ± 4%, respectively). Maturation for all vitrified-warmed oocyte treatments (37 ± 6.5 to 45.9 ± 5.8%) was not different from control (50 ± 4.1%), except for MAT CR- CO₂ (20.3 ± 4.6%). Cleavage for MAT CR- CO₂ and Air groups was similar to control (67.7 ± 12.1, 71.4 ± 8.1, and 78 ± 5.3%, respectively). One blastocyst was produced (MAT CR + CO₂), representing the first equine blastocyst reported after vitrification of an in vitro-matured oocyte.