Richard G. Vogt

The insect SNMP gene family.

SNMPs are membrane proteins observed to associate with chemosensory neurons in insects; in Drosophila melanogaster, SNMP1 has been shown to be essential for the detection of the pheromone cis-vaccenyl acetate (CVA). SNMPs are one of three insect gene clades related to the human fatty acid transporter CD36. We previously characterized the CD36 gene family in 4 insect Orders that effectively cover the Holometabola, or some 80% of known insect species and the 300 million years of evolution since this lineage emerged: Lepidoptera (e.g. Bombyx mori, Antheraea polyphemus, Manduca sexta, Heliothis virescens, Helicoverpa assulta, Helicoverpa armigera, Mamestra brassicae); Diptera (D. melanogaster, Drosophila pseudoobscura, Aedes aegypti, Anopheles gambiae, Culex pipiens quinquefasciatus); Hymenoptera (Apis mellifera); and Coleoptera (Tribolium castaneum). This previous study suggested a complex topography within the SNMP clade including a strongly supported SNMP1 sub-clade plus additional SNMP genes. To further resolve the SNMP clade here, we used cDNA sequences of SNMP1 and SNMP2 from various Lepidoptera species, D. melanogaster and Ae. aegypti, as well as BAC derived genomic sequences from Ae. aegypti as models for proposing corrected sequences of orthologues in the D. pseudoobscura and An. gambiae genomes, and for identifying orthologues in the B. mori and C. pipiens q. genomes. We then used these sequences to analyze the SNMP clade of the insect CD36 gene family, supporting the existence of two well supported sub-clades, SNMP1 and SNMP2, throughout the dipteran and lepidopteran lineages, and plausibly throughout the Holometabola and across a broad evolutionary time scale. We present indirect evidence based on evolutionary selection (dN/dS) that the dipteran SNMPs are expressed as functional proteins. We observed expansions of the SNMP1 sub-clade in C. pipiens q. and T. castaneum suggesting that the SNMP1s may have an expanded functional role in these species.

The SNMP/CD36 gene family in Diptera, Hymenoptera and Coleoptera: Drosophila melanogaster, D. pseudoobscura, Anopheles gambiae, Aedes aegypti, Apis mellifera, and Tribolium castaneum.

Sensory neuron membrane proteins (SNMPs) are membrane bound proteins initially identified in olfactory receptor neurons of Lepidoptera and are thought to play a role in odor detection; SNMPs belong to a larger gene family characterized by the human protein CD36. We have identified 12-14 candidate SNMP/CD36 homologs from each of the genomes of Drosophila melanogaster, D. pseudoobscura, Anopheles gambiae and Aedes aegypti (Diptera), eight candidate homologs from Apis mellifera (Hymenoptera), and 15 from Tribolium castaneum (Coleoptera). Analysis (sequence similarity and intron locations) suggests that the insect SNMP/CD36 genes fall into three major groups. Group 1 includes the previously characterized D. melanogaster emp (epithelial membrane protein). Group 2 includes the previously characterized D. melanogaster croquemort, ninaD, santa maria, and peste. Group 3 genes include the SNMPs, which fall into two subgroups referred to as SNMP1 and SNMP2. D. melanogaster SNMP1 (CG7000) shares both significant sequence similarity and five of its six intron insertion sites with the lepidopteran Bombyx mori SNMP1. The topological conservation of this gene family within the three major holometabolous lineages indicates that it predates the coleopteran and hymenoptera/dipera/lepidoptera split 300+ million years ago. The current state of knowledge of the characterized insect members of this gene family is discussed.

Odorant Binding Protein Homologues of the Malaria Mosquito Anopheles gambiae; Possible Orthologues of the OS-E and OS-F OBPs of Drosophila melanogaster

Twenty-nine Anopheles gambiae candidate Odorant Binding Proteins (OBPs) were characterized for similarity to OBPs of Drosophila melanogaster and other insects. Twenty-five of these sequences were identified by BLAST searching the A. gambiae genome database. Several A. gambiae sequences were significantly similar to the D. melanogaster OBPs OS-E/OS-F, LUSH and PBPRP2/PBPRP5. Exon boundary comparisons suggests that two A. gambiae genes are orthologues of OS-E and OS-F, justifying the names AgamOS-E (EAA01090, AF437886) and AgamOS-F (EAA14641, AF437884). If these are orthologues, then the gene duplication establishing the OS-E and OS-F lineages predated the divergence of mosquitoes and flies. The identification of orthologous OBPs and other chemosensory genes between D. melanogaster and A. gambiae should accelerate the transfer of physiological and behavioral information between these two species.

Expression of SNMP-1 in olfactory neurons and sensilla of male and female antennae of the silkmoth Antheraea polyphemus

Abstract. SNMP-1 (sensory neuron membrane protein 1) is an olfactory-specific membrane-bound protein which is homologous with the CD36 receptor family. Previous light level immunocytochemical studies suggested that SNMP-1 was localized in the dendrites and distal cell body of sex-pheromone-specific olfactory receptor neurons (ORN); these studies further suggested SNMP-1 was expressed in only one of two to three neurons in male-specific pheromone-sensitive trichoid sensilla. To better understand the expression and localization of SNMP-1, an immunocytochemical study was performed using electron microscopy to visualize the distribution of SNMP-1 among the neurons of several classes of olfactory sensilla of both male and female antennae of the silkmoth Antheraea polyphemus. SNMP-1 antigenicity was primarily restricted to the receptive dendritic membranes of ORNs of all sensilla types examined and was observed in cytosolic granules, but not plasma membranes, of the cell soma. Mean labeling densities ranged from 1 to 16 gold particles per micrometer of dendrite circumference; dendrites of trichoid and intermediate sensilla showed significantly higher labeling densities than those of basiconic sensilla. Larger dendrites of trichoid sensilla showed significantly higher mean labeling densities (13–16/µm) than smaller diameter dendrites (3–7/µm). Immunofluorescence studies using baculovirus expressed SNMP-1 and multiphoton photon laser scanning microscopy (MPLSM) indicated that rSNMP-1, which was post-translationally processed to the in vivo molecular weight, was inserted into the plasma membrane in a topography presenting extracellular epitopes. These studies suggest SNMP-1 is a common feature of the ORNs, is asymmetrically expressed among functionally distinct neurons, and possesses a topography which permits interaction with components of the extracellular sensillum lymph.

Ontogeny of odorant receptor gene expression in zebrafish, Danio rerio

We cloned three putative odorant receptor (OR) genes from the zebrafish to use as in situ hybridization probes to follow the temporal patterns of neurons expressing OR genes through a developmental progression from embryo (12 h postfertilization) to adult. The identification of these genes is supported by sequence homology to previously reported ORs and by the morphology and location of labeled cells in in situ hybridization experiments. Cells expressing OR mRNA were first observed in the olfactory placodes between 31 and 38 h after fertilization (fish reared at 26 degrees C). Initially, only single cells were observed to hybridize the probe; the number of labeled cells increased throughout the remainder of embryogenesis and through postembryonic growth and morphogenesis of the olfactory organ. At all ages, the positively hybridizing cells were scattered throughout the olfactory epithelium but not in the nonsensory epithelium of the olfactory organ.

High level expression of “male specific” pheromone binding proteins (PBPs) in the antennae of female noctuiid moths

Pheromone Binding Proteins (PBPs) are one branch of a multigene family of lepidopteran Odorant Binding Proteins (OBPs) that are known for their relatively high levels of expression in male antennae. However, PBP expression has been observed at low levels in female antennae of the Saturniidae, Bombycidae and Lymantriidae, and at relatively high levels in members of the Noctuiidae. The function of female PBP expression is unclear, as female lepidoptera are consistently noted for their failure to respond physiologically or behaviorally to sex-pheromone. In this study, the sexual dimorphism of PBP expression was examined in the noctuiid moths Helicoverpa zea, Heliothis virescens and Spodoptera frugiperda. A PBP cDNA clone was isolated from female H. zea, PBP-Hzea(f). Northern blot analysis indicated relatively high levels of PBP-Hzea(f) expression in both male and female antennae, though females consistently expressed about 50% that of males. Western blot analysis of male and female PBP expression supported these relative differences. Immunocytochemical analysis indicates discrete expression localized beneath olfactory sensilla of both male and female antennae. These results suggest female noctuiids possess the biochemistry to detect at least components of their sex-pheromone. Alternatively, these results may suggest that PBPs have a more general function in noctuiids, possibly reflecting behavioral and life history differences that distinguish this the Noctuiidae from other Lepidopteran families.

PATTERNS OF GENE DUPLICATION IN LEPIDOPTERAN PHEROMONE BINDING PROTEINS

Abstract. We have isolated and characterized cDNAs representing two distinct pheromone binding proteins (PBPs) from the gypsy moth, Lymantria dispar. We use the L. dispar protein sequences, along with other published lepidopteran PBPs, to investigate the evolutionary relationships among genes within the PBP multigene family. Our analyses suggest that the presence of two distinct PBPs in genera representing separate moth superfamilies is the result of relatively recent, independent, gene duplication events rather than a single, ancient, duplication. We discuss this result with respect to the biochemical diversification of moth PBPs.

The Lepidoptera Odorant Binding Protein gene family: Gene gain and loss within the GOBP/PBP complex of moths and butterflies

Butterflies and moths differ significantly in their daily activities: butterflies are diurnal while moths are largely nocturnal or crepuscular. This life history difference is presumably reflected in their sensory biology, and especially the balance between the use of chemical versus visual signals. Odorant Binding Proteins (OBP) are a class of insect proteins, at least some of which are thought to orchestrate the transfer of odor molecules within an olfactory sensillum (olfactory organ), between the air and odor receptor proteins (ORs) on the olfactory neurons. A Lepidoptera specific subclass of OBPs are the GOBPs and PBPs; these were the first OBPs studied and have well documented associations with olfactory sensilla. We have used the available genomes of two moths, Manduca sexta and Bombyx mori, and two butterflies, Danaus plexippus and Heliconius melpomene, to characterize the GOBP/PBP genes, attempting to identify gene orthologs and document specific gene gain and loss. First, we identified the full repertoire of OBPs in the M. sexta genome, and compared these with the full repertoire of OBPs from the other three lepidopteran genomes, the OBPs of Drosophila melanogaster and select OBPs from other Lepidoptera. We also evaluated the tissue specific expression of the M. sexta OBPs using an available RNAseq databases. In the four lepidopteran species, GOBP2 and all PBPs reside in single gene clusters; in two species GOBP1 is documented to be nearby, about 100 kb from the cluster; all GOBP/PBP genes share a common gene structure indicating a common origin. As such, the GOBP/PBP genes form a gene complex. Our findings suggest that (1) the lepidopteran GOBP/PBP complex is a monophyletic lineage with origins deep within Lepidoptera phylogeny, (2) within this lineage PBP gene evolution is much more dynamic than GOBP gene evolution, and (3) butterflies may have lost a PBP gene that plays an important role in moth pheromone detection, correlating with a shift from olfactory (moth) to visual (butterfly) communication, at least regarding long distance mate recognition. These findings will be clarified by additional lepidopteran genomic data, but the observation that moths and butterflies share most of the PBP/GOBP genes suggests that they also share common chemosensory-based behavioral pathways.

Scale esterase : A pheromone-degrading enzyme from scales of silk mothAntheraea polyphemus.

Body scales of the silk mothAntheraea polyphemus contain an esterase which can degrade the female sex pheromone of this species. This esterase, which appears to be stabilized to the scale cuticle, is present in both sexes, but is species specific. The enzyme may play a significant role in the behaviors associated with sex-pheromone attraction, helping to filter out stimulus noise by degrading adsorbed pheromone, thus preventing adsoptive body surfaces from becoming uncontrolled pheromone sources.

Infection of lepidoptera with a pseudotyped retroviral vector

Studies requiring the introduction and expression of manipulated gene constructs have been technically difficult in non-drosophilid insects. Retroviruses can be engineered to be replication defective and to serve as vectors for gene constructs of interest. In this study, pseudotyped MoMLV(VSV-G) retroviral vectors are shown to successfully infect lepidopteran cells in vitro and in vivo. In Spodoptera frugiperda cells in vitro and in Manduca sexta in vivo, infection and conversion to proviral DNA were confirmed by PCR amplification and Southern blot hybridization of vector-specific sequences. Gene expression and integration of proviral DNA were also documented in vitro. This is the first report of retroviral infection in lepidoptera and suggests that pseudotyped retroviral vectors could be powerful tools in gene manipulation studies of non-drosophilid insects.