Neuropeptides and peptide hormones identified in codling moth, Cydia pomonella (Lepidoptera: Tortricidae).

The codling moth, Cydia pomonella, is a worldwide pest of pome fruits. Neuropeptides regulate most physiological functions in insects and represent new targets for the development of control agents. The only neuropeptides reported from the codling moth to date are the allatostatin A family peptides. To identify other neuropeptides and peptide hormones from codling moth, we analyzed head transcriptomes, identified 50 transcripts, and predicted 120 prepropeptides for the codling moth neuropeptides and peptide hormones. All transcripts were amplified, and these sequences were verified. One of the notable findings in this study is that diapause hormones (DHs) reported from Tortricid moths, including the codling moth, do not have the WFGPRL sequence in C-terminal ends in the pban genes. The C-terminal motif is critical to characterize insect DH peptides, and always conserved in pban/dh genes in Lepidoptera and many insect orders. Interestingly, the WFGPRL sequence was produced only from the capa gene in the codling moth. The allatostatin A-family encoding transcript predicted nine peptides, seven of which, as expected, are identical to those previously isolated from the moth. We also identified new codling moth orthologs of insect neuropeptides including CCHamides, allatostatin CC, RYamides, and natalisins. The information provided in this study will benefit future codling moth investigations using peptidoproteomics to determine peptide presence and functions.

CRISPR/Cas9 Editing of the Codling Moth (Lepidoptera: Tortricidae) CpomOR1 Gene Affects Egg Production and Viability.

The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), is a major pest of pome fruit worldwide. Incorporation of semiochemicals, including the main sex pheromone (codlemone), into codling moth IPM programs has drastically reduced the amount of chemical insecticides needed to control this orchard pest. Odorant receptors located in sensory neuron membranes in the antennae are key sensors in the detection of semiochemicals and trigger downstream signaling events leading to a behavioral response. CpomOR1 is an odorant receptor belonging to the pheromone receptor subfamily in codling moth, and is a prime candidate for being a codlemone receptor based on its high expression levels in male antennae. In this study, the CpomOR1 gene was targeted using CRISPR/Cas9 genome editing to knockdown functional OR1 protein production to determine physiological function(s). By injecting early stage eggs, mutations were successfully introduced, including both deletions and insertions. When attempting to create stable populations of codling moth through mating of males with females containing mutations of the CpomOR1 gene, it was found that fecundity and fertility were affected, with edited females producing nonviable eggs. The role of CpomOR1 in fecundity and fertility in codling moth is unknown and will be the focus of future studies.

Bacterial Endosymbionts of the Psyllid Cacopsylla pyricola (Hemiptera: Psyllidae) in the Pacific Northwestern United States.

Insects often have facultative associations with bacterial endosymbionts, which can alter the insects' susceptibility to parasitism, pathogens, plant defenses, and certain classes of insecticides. We collected pear psylla, Cacopsylla pyricola (Förster) (Hemiptera: Psyllidae), from pear orchards in Washington and Oregon, and surveyed them for the presence of bacterial endosymbionts. Adult psyllids were collected on multiple dates to allow us to assay specimens of both the summer ("summerform") and the overwintering ("winterform") morphotypes. Two endosymbionts, Arsenophonus and Phytoplasma pyri, were detected in psyllids of both morphotypes in both states. A separate survey revealed similar associations present in psyllids collected in 1987. Arsenophonus was present in 80-100% of psyllids in all growing regions. A slightly lower proportion of summerform than winterform psyllids harbored the bacterium. Arsenophonus was present in the bacteriomes and developing oocytes of most psyllids, indicating that this endosymbiont is transovarially transmitted. This bacterium was also observed in the salivary glands and midguts of some psyllids. Phytoplasma pyri was present in a greater proportion of pear psylla from orchards near Yakima, WA, than from other regions, and was present in a higher proportion of winterforms than summerforms. We did not detect Wolbachia, Profftella, or Liberibacter europaeus, which are associated with other psyllid pests, including other species of Cacopsylla. Our study is the first to survey North American populations of C. pyricola for endosymbionts, and provides a foundation for further research on how bacterial associations may influence the ecology and management of this pest.

Gut Content Analysis of a Phloem-Feeding Insect, Bactericera cockerelli (Hemiptera: Triozidae).

Potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), is a key pest of potato (Solanum tuberosum L., Solanales: Solanaceae) and a vector of "Candidatus Liberibacter solanacearum," the pathogen associated with zebra chip disease. In addition to its presence on cultivated crops, the psyllid regularly occurs on numerous uncultivated annual and perennial species within the Solanaceae. A better understanding of landscape-level ecology of B. cockerelli would substantially improve our ability to predict which potato fields are most likely to be colonized by infected psyllids. We developed three PCR-based methods of gut content analysis to identify what plant species B. cockerelli had previously fed upon. These methods included-1) sequencing PCR amplicons of regions of plant-derived internal transcribed spacer (ITS) or the chloroplast trnL gene from psyllids, 2) high-resolution melting analysis of ITS or trnL real-time PCR products, and 3) restriction enzyme digestion of trnL PCR product. Each method was used to test whether we could identify psyllids that had been reared continuously on potato versus psyllids reared continuously on the perennial nightshade, Solanum dulcamara. All three methods of gut content analysis correctly identified psyllids from potato and psyllids from S. dulcamara Our study is the first to demonstrate that plant DNA can be detected in a phloem-feeding insect. Gut content analysis, in combination with other landscape ecology approaches, could help elucidate patterns in landscape-level movements and host plant associations of B. cockerelli.

Releases of insectary-reared Galendromus occidentalis (Acari: Phytoseiidae) in commercial apple orchards.

Galendromus occidentalis (Nesbitt) is one of several phytoseiid species that are available for purchase to supplement endemic predator populations that are not providing sufficient control of spider mites. We performed a series of releases of commercially reared G. occidentalis in commercial apple (Malus domestica Borkhausen) orchards in Washington from 2010 to 2012. Releases of up to 50,000 mites per acre did not lead to an increase in populations of predatory mites or to a decrease in populations of pest mites. Assessments of mite numbers in shipments and quality (survival and fecundity) of those mites indicated that the commercial insectary was correctly estimating the number of predatory mites in their shipments, and that predator quality was not different than a laboratory colony. Finally, a predator-prey model that used the intrinsic rates of increase of tetranychid prey and the prey consumption rate of the predator indicated that the density of G. occidentalis required to control the prey at the action threshold was not economically feasible. We conclude that G. occidentalis cannot be used to bring about short-term control via inundative releases in crops such as apple with large canopy volumes.

Application of Cydia pomonella expressed sequence tags: Identification and expression of three general odorant binding proteins in codling moth.

The codling moth, Cydia pomonella, is one of the most important pests of pome fruits in the world, yet the molecular genetics and the physiology of this insect remain poorly understood. A combined assembly of 8 341 expressed sequence tags was generated from Roche 454 GS-FLX sequencing of eight tissue-specific cDNA libraries. Putative chemosensory proteins (12) and odorant binding proteins (OBPs) (18) were annotated, which included three putative general OBP (GOBP), one more than typically reported for other Lepidoptera. To further characterize CpomGOBPs, we cloned cDNA copies of their transcripts and determined their expression patterns in various tissues. Cloning and sequencing of the 698 nt transcript for CpomGOBP1 resulted in the prediction of a 163 amino acid coding region, and subsequent RT-PCR indicated that the transcripts were mainly expressed in antennae and mouthparts. The 1 289 nt (160 amino acid) CpomGOBP2 and the novel 702 nt (169 amino acid) CpomGOBP3 transcripts are mainly expressed in antennae, mouthparts, and female abdomen tips. These results indicate that next generation sequencing is useful for the identification of novel transcripts of interest, and that codling moth expresses a transcript encoding for a new member of the GOBP subfamily.

Survey of resistance to four insecticides and their associated mechanisms in different genotypes of the green peach aphid (Hemiptera: Aphididae) from Chile.

The green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae) is amajor pest of agriculture worldwide that has proved to be particularly adept at evolving insecticide resistance. Several mechanisms that confer resistance to many insecticide types have been described in M. persicae. We measured the resistance status of nine multilocus genotypes (MLGs) of this aphid species collected in Chile. MLGs were identified using microsatellite markers, and these MLG clonal populations were measured for the presence of modified acetylcholinesterase (MACE), kdr and super kdr mutations, and enhanced carboxyl esterase activity. Toxicological bioassays were used to estimate aphid LC50 when treated with metamidophos (organophosphate), pirimicarb (dimethyl carbamate), cyfluthrin (pyrethroid), and imidacloprid (neonicotinoid). Two MLGs presented >20-fold resistance to pirimicarb, which was associated with the MACE mutation in the heterozygous condition. The kdr mutation was found in only four MLGs in the heterozygous condition and they showed resistance ratios (RR) to cyfluthrin of less than sevenfold. The super kdr mutation was not detected. Enhanced carboxyl esterase activity was predominantly found in the susceptible (S) to first level of resistance (R1) with RR to metamidophos less than eight-fold. Finally, RR to imidacloprid was also less than eight-fold in all MLGs tested. A few MLGs with resistance to pirimicarb were found, while susceptibility to cyfluthrin, metamidophos and imidacloprid was still predominant. A significant positive correlation between imidacloprid tolerance with pirimicarb resistance was detected, as well as between imidacloprid and metamidophos tolerance. With the increase in the use of neonicotinoid insecticides, better rotation of insecticides with different modes of action will be necessary to prevent further development of M. persicae insecticide resistance in Chile.

Tree fruit IPM programs in the western United States: the challenge of enhancing biological control through intensive management.

The seminal work of Stern and his coauthors on integrated control has had a profound and long-lasting effect on the development of IPM programs in western orchard systems. Management systems based solely on pesticides have proven to be unstable, and the success of IPM systems in western orchards has been driven by conservation of natural enemies to control secondary pests, combined with pesticides and mating disruption to suppress the key lepidopteran pests. However, the legislatively mandated changes in pesticide use patterns prompted by the Food Quality Protection Act of 1996 have resulted in an increased instability of pest populations in orchards because of natural enemy destruction. The management system changes have made it necessary to focus efforts on enhancing biological control not only of secondary pests but also of primary lepidopteran pests to help augment new pesticides and mating disruption tactics. The new management programs envisioned will be information extensive as well as time sensitive and will require redesign of educational and outreach programs to be successful. The developing programs will continue to use the core principles of Stern and his co-authors, but go beyond them to incorporate changes in society, technology and information transfer, as needed.

Interactions of two idiobiont parasitoids (Hymenoptera: Ichneumonidae) of codling moth (Lepidoptera: Tortricidae) with the entomopathogenic nematode Steinernema carpocapsae (Rhabditida: Steinernematidae).

Simultaneous use of parasitoids and entomopathogenic nematodes for codling moth (CM) control could produce an antagonistic interaction between the two groups resulting in death of the parasitoid larvae. Two ectoparasitic ichneumonid species, Mastrus ridibundus and Liotryphon caudatus, imported for classical biological control of cocooned CM larvae were studied regarding their interactions with Steinernema carpocapsae. Exposure of M. ridibundus and L. caudatus developing larvae to infective juveniles (IJs) of S. carpocapsae (10 IJs/cm2; approximately LC(80-90) for CM larvae) within CM cocoons resulted in 70.7 and 85.2% mortality, respectively. However, diapausing full grown parasitoid larvae were almost completely protected from nematode penetration within their own tightly woven cocoons. M. ridibundus and L. caudatus females were able to detect and avoid ovipositing on nematode-infected cocooned CM moth larvae as early as 12h after treatment of the host with IJs. When given the choice between cardboard substrates containing untreated cocooned CM larvae and those treated with an approximate LC95 of S. carpocapsae IJs (25 IJs/cm2) 12, 24, or 48h earlier, ovipositing parasitoids demonstrated a significant preference for untreated larvae. The ability of these parasitoids to avoid nematode-treated larvae and to seek out and kill cocooned CM larvae that survive nematode treatments enhances the complementarity of entomopathogenic nematodes and M. ridibundus and L. caudatus.