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Background: Lucilia cuprina (Wiedemann, 1830) (Diptera: Calliphoridae) is the main causative agent of flystrike of sheep in Australia and New Zealand. Female flies lay eggs in an open wound or natural orifice, and the developing larvae eat the host's tissues, a condition called myiasis. To improve our understanding of host-seeking behavior, we quantified gene expression in male and female antennae based on their behavior., Methods: A spatial olfactometer was used to evaluate the olfactory response of L. cuprina mated males and gravid females to fresh or rotting beef. Antennal RNA-Seq analysis was used to identify sensory receptors differentially expressed between groups., Results: Lucilia cuprina females were more attracted to rotten compared to fresh beef (> fivefold increase). However, males and some females did not respond to either type of beef. RNA-Seq analysis was performed on antennae dissected from attracted females, non-attracted females and males. Transcripts encoding sensory receptors from 11 gene families were identified above a threshold (≥ 5 transcript per million) including 49 ATP-binding cassette transporters (ABCs), two ammonium transporters (AMTs), 37 odorant receptors (ORs), 16 ionotropic receptors (IRs), 5 gustatory receptors (GRs), 22 odorant-binding proteins (OBPs), 9 CD36-sensory neuron membrane proteins (CD36/SNMPs), 4 chemosensory proteins (CSPs), 4 myeloid lipid-recognition (ML) and Niemann-Pick C2 disease proteins (ML/NPC2), 2 pickpocket receptors (PPKs) and 3 transient receptor potential channels (TRPs). Differential expression analyses identified sex-biased sensory receptors., Conclusions: We identified sensory receptors that were differentially expressed between the antennae of both sexes and hence may be associated with host detection by female flies. The most promising for future investigations were as follows: an odorant receptor (LcupOR46) which is female-biased in L. cuprina and Cochliomyia hominivorax Coquerel, 1858; an ABC transporter (ABC G23.1) that was the sole sensory receptor upregulated in the antennae of females attracted to rotting beef compared to non-attracted females; a female-biased ammonia transporter (AMT_Rh50), which was previously associated with ammonium detection in Drosophila melanogaster Meigen, 1830. This is the first report suggesting a possible role for ABC transporters in L. cuprina olfaction and potentially in other insects., (© 2024. The Author(s).)

Background: Diachasmimorpha longicaudata is a hymenopteran fruit fly endoparasitoid. Females of this species find their hosts for oviposition by using complex sensorial mechanisms in response to physical and chemical stimuli associated with the host and host habitat. Ecological and behavioral aspects related to host-seeking behavior for oviposition have been extensively studied in D. longicaudata, including the identification of volatile organic compounds acting as attractants to females. In this sense, molecular mechanisms of chemoreception have been explored in this species, including a preliminary characterization of odorant-binding proteins (OBPs), chemosensory proteins (CSPs) and odorant receptors (ORs), among other proteins. Functional assays on OBP and CSP have been conducted as a first approach to identify molecular mechanisms associated with the female host-seeking behavior for oviposition. The aims of the present study were to identify the D. longicaudata sensory gene repertoire expressed in the antenna of sexually mature and mated individuals of both sexes, and subsequently, characterize transcripts differentially expressed in the antennae of females to identify candidate genes associated with the female host-seeking behavior for oviposition., Results: A total of 33,745 predicted protein-coding sequences were obtained from a de novo antennal transcriptome assembly. Ten sensory-related gene families were annotated as follows: 222 ORs, 44 ionotropic receptors (IRs), 25 gustatory receptors (GRs), 9 CSPs, 13 OBPs, 2 ammonium transporters (AMTs), 8 pickpocket (PPKs) receptors, 16 transient receptor potential (TRP) channels, 12 CD36/SNMPs and 3 Niemann-Pick type C2 like proteins (NPC2-like). The differential expression analysis revealed 237 and 151 transcripts up- and downregulated, respectively, between the female and male antennae. Ninety-seven differentially expressed transcripts corresponded to sensory-related genes including 88 transcripts being upregulated (87 ORs and one TRP) and nine downregulated (six ORs, two CSPs and one OBP) in females compared to males., Conclusions: The sensory gene repertoire of D. longicaudata was similar to that of other taxonomically related parasitoid wasps. We identified a high number of ORs upregulated in the female antenna. These results may indicate that this gene family has a central role in the chemoreception of sexually mature females during the search for hosts and host habitats for reproductive purposes., (© 2024. The Author(s).)

Background: The cattle fever tick, Rhipicephalus (Boophilus) microplus, is a vector of pathogens causative of babesiosis and anaplasmosis, both highly lethal bovine diseases that affect cattle worldwide. In Ecdysozoa, neuropeptides and their G-protein-coupled receptors play a critical integrative role in the regulation of all physiological processes. However, the physiological activity of many neuropeptides is still unknown in ticks. Periviscerokinins (CAP 2b /PVKs) are neuropeptides associated with myotropic and diuretic activities in insects. These peptides have been identified only in a few tick species, such as Ixodes ricinus, Ixodes scapularis and R. microplus, and their cognate receptor only characterized for the last two., Methods: Expression of the periviscerokinin receptor (Rhimi-CAP 2b R) was investigated throughout the developmental stages of R. microplus and silenced by RNA interference (RNAi) in the females. In a first experiment, three double-stranded (ds) RNAs, named ds680-805, ds956-1109 and ds1102-1200, respectively, were tested in vivo. All three caused phenotypic effects, but only the last one was chosen for subsequent experiments. Resulting RNAi phenotypic variables were compared to those of negative controls, both non-injected and dsRNA beta-lactamase-injected ticks, and to positive controls injected with beta-actin dsRNA. Rhimi-CAP 2b R silencing was verified by quantitative reverse-transcriptase PCR in whole females and dissected tissues., Results: Rhimi-CAP 2b R transcript expression was detected throughout all developmental stages. Rhimi-CAP 2b R silencing was associated with increased female mortality, decreased weight of surviving females and of egg masses, a delayed egg incubation period and decreased egg hatching (P < 0.05)., Conclusions: CAP 2b /PVKs appear to be associated with the regulation of female feeding, reproduction and survival. Since the Rhimi-CAP 2b R loss of function was detrimental to females, the discovery of antagonistic molecules of the CAP 2b /PVK signaling system should cause similar effects. Our results point to this signaling system as a promising target for tick control., (© 2022. The Author(s).)

In ecdysozoan animals, moulting entails the production of a new exoskeleton and shedding of the old one during ecdysis. It is induced by a pulse of ecdysone that regulates the expression of different hormonal receptors and activates a peptide-mediated signalling cascade. In Holometabola, the peptidergic cascade regulating ecdysis has been well described. However, very little functional information regarding the neuroendocrine regulation of ecdysis is available for Hemimetabola, which display an incomplete metamorphosis. We use Rhodnius prolixus as a convenient experimental model to test two hypotheses: (1) the role of neuropeptides that regulate ecdysis in Holometabola is conserved in hemimetabolous insects; and (2) the neuropeptides regulating ecdysis play a role in the regulation of female reproduction during the adult stage. The RNA interference-mediated reduction of ecdysis triggering hormone (ETH) mRNA levels in fourth-instar nymphs resulted in lethality at the expected time of ecdysis. Unlike in holometabolous insects, knockdown of eth and orcokinin isoform A (oka) did not affect oviposition in adult females, pointing to a different endocrine regulation of ovary maturation. However, eth knockdown prevented egg hatching. The blockage of egg hatching appears to be a consequence of embryonic ecdysis failure. Most of the first-instar nymphs hatched from the eggs laid by females injected with dsRNA for eclosion hormone (dsEH), crustacean cardioactive peptide (dsCCAP) and dsOKA died at the expected time of ecdysis, indicating the crucial involvement of these genes in post-embryonic development. No phenotypes were observed upon corazonin (cz) knockdown in nymphs or adult females. The results are relevant for evolutionary entomology and could reveal targets for neuropeptide-based pest control tools., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Background: Rhipicephalus microplus is the vector of deadly cattle pathogens, especially Babesia spp., for which a recombinant vaccine is not available. Therefore, disease control depends on tick vector control. However, R. microplus populations worldwide have developed resistance to available acaricides, prompting the search for novel acaricide targets. G protein-coupled receptors (GPCRs) are involved in the regulation of many physiological processes and have been suggested as druggable targets for the control of arthropod vectors. Arthropod-specific signaling systems of small neuropeptides are being investigated for this purpose. The pyrokinin receptor (PKR) is a GPCR previously characterized in ticks. Myotropic activity of pyrokinins in feeding-related tissues of Rhipicephalus sanguineus and Ixodes scapularis was recently reported., Methods: The R. microplus pyrokinin receptor (Rhimi-PKR) was silenced through RNA interference (RNAi) in female ticks. To optimize RNAi, a dual-luciferase assay was applied to determine the silencing efficiency of two Rhimi-PKR double-stranded RNAs (dsRNA) prior to injecting dsRNA in ticks to be placed on cattle. Phenotypic variables of female ticks obtained at the endpoint of the RNAi experiment were compared to those of control female ticks (non-injected and beta-lactamase dsRNA-injected). Rhimi-PKR silencing was verified by quantitative reverse-transcriptase PCR in whole females and dissected tissues., Results: The Rhimi-PKR transcript was expressed in all developmental stages. Rhimi-PKR silencing was confirmed in whole ticks 4 days after injection, and in the tick carcass, ovary and synganglion 6 days after injection. Rhimi-PKR silencing was associated with an increased mortality and decreased weight of both surviving females and egg masses (P < 0.05). Delays in repletion, pre-oviposition and incubation periods were observed (P < 0.05)., Conclusions: Rhimi-PKR silencing negatively affected female reproductive fitness. The PKR appears to be directly or indirectly associated with the regulation of female feeding and/or reproductive output in R. microplus. Antagonists of the pyrokinin signaling system could be explored for tick control., (© 2022. The Author(s).)

Neuropeptides regulate many important physiological processes in animals. The G protein-coupled receptors of corresponding small neuropeptide ligands are considered promising targets for controlling arthropod pests. Pyrokinins (PKs) are pleiotropic neuropeptides that, in some insect species, stimulate muscle contraction and modulate pheromone biosynthesis, embryonic diapause, and feeding behavior. However, their function remains unknown in ticks. In this study, we reported the myotropic activity of tick endogenous PKs and a PK agonist analog, PK-PEG 8 (MS[PEG 8 ]-YFTPRLa), on feeding tissues of two tick species representing the family Ixodidae lineages, namely, Prostriata ( Ixodes scapularis ) and Metastriata ( Rhipicephalus sanguineus ). First, we predicted the sequences of two periviscerokinins (PVK), one with a derived ending RNa and five PKs encoded by the CAPA peptide precursor from R. sanguineus and found the encoded PKs were identical to those of R. microplus identified previously. The pharynx-esophagus of both tick species responded with increased contractions to 10 μM of the endogenous PK as well as to PK-PEG 8 but not to the scrambled PK peptide, as expected. A dose-dependent myotropic activity of the PK-PEG 8 was found for both tick species, validating the analog activity previously found in the pyrokinin recombinant receptor assay. In agreement with the tissue activity elicited, we quantified the relative transcript abundance of R. sanguineus PK receptor in unfed female ticks and found it was the highest in the feeding tissues extracted from the capitulum and lowest in the reproductive tissue. This is the first report of the activity of pyrokinins in ticks. These findings strongly indicate the potential role of PKs in regulating tick blood feeding and therefore, making the tick PK receptor a potential target for interference., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xiong, Wulff, Nachman and Pietrantonio.)