BackgroundBed bugs (Cimex lectularius) are hematophagous nocturnal parasites of humans that have attained high impact status due to their worldwide resurgence. The sudden and rampant resurgence of C. lectularius has been attributed to numerous factors including frequent international travel, narrower pest management practices, and insecticide resistance.ResultsWe performed a next-generation RNA sequencing (RNA-Seq) experiment to find differentially expressed genes between pesticide-resistant (PR) and pesticide-susceptible (PS) strains of C. lectularius. A reference transcriptome database of 51,492 expressed sequence tags (ESTs) was created by combining the databases derived from de novo assembled mRNA-Seq tags (30,404 ESTs) and our previous 454 pyrosequenced database (21,088 ESTs). The two-way GLMseq analysis revealed ~15,000 highly significant differentially expressed ESTs between the PR and PS strains. Among the top 5,000 differentially expressed ESTs, 109 putative defense genes (cuticular proteins, cytochrome P450s, antioxidant genes, ABC transporters, glutathione S-transferases, carboxylesterases and acetyl cholinesterase) involved in penetration resistance and metabolic resistance were identified. Tissue and development-specific expression of P450 CYP3 clan members showed high mRNA levels in the cuticle, Malpighian tubules, and midgut; and in early instar nymphs, respectively. Lastly, molecular modeling and docking of a candidate cytochrome P450 (CYP397A1V2) revealed the flexibility of the deduced protein to metabolize a broad range of insecticide substrates including DDT, deltamethrin, permethrin, and imidacloprid.ConclusionsWe developed significant molecular resources for C. lectularius putatively involved in metabolic resistance as well as those participating in other modes of insecticide resistance. RNA-Seq profiles of PR strains combined with tissue-specific profiles and molecular docking revealed multi-level insecticide resistance in C. lectularius. Future research that is targeted towards RNA interference (RNAi) on the identified metabolic targets such as cytochrome P450s and cuticular proteins could lay the foundation for a better understanding of the genetic basis of insecticide resistance in C. lectularius.

BackgroundBed bugs (Cimex lectularius) are blood-feeding insects poised to become one of the major pests in households throughout the United States. Resistance of C. lectularius to insecticides/pesticides is one factor thought to be involved in its sudden resurgence. Despite its high-impact status, scant knowledge exists at the genomic level for C. lectularius. Hence, we subjected the C. lectularius transcriptome to 454 pyrosequencing in order to identify potential genes involved in pesticide resistance.Methodology and Principal FindingsUsing 454 pyrosequencing, we obtained a total of 216,419 reads with 79,596,412 bp, which were assembled into 35,646 expressed sequence tags (3902 contigs and 31744 singletons). Nearly 85.9% of the C. lectularius sequences showed similarity to insect sequences, but 44.8% of the deduced proteins of C. lectularius did not show similarity with sequences in the GenBank non-redundant database. KEGG analysis revealed putative members of several detoxification pathways involved in pesticide resistance. Lamprin domains, Protein Kinase domains, Protein Tyrosine Kinase domains and cytochrome P450 domains were among the top Pfam domains predicted for the C. lectularius sequences. An initial assessment of putative defense genes, including a cytochrome P450 and a glutathione-S-transferase (GST), revealed high transcript levels for the cytochrome P450 (CYP9) in pesticide-exposed versus pesticide-susceptible C. lectularius populations. A significant number of single nucleotide polymorphisms (296) and microsatellite loci (370) were predicted in the C. lectularius sequences. Furthermore, 59 putative sequences of Wolbachia were retrieved from the database.ConclusionsTo our knowledge this is the first study to elucidate the genetic makeup of C. lectularius. This pyrosequencing effort provides clues to the identification of potential detoxification genes involved in pesticide resistance of C. lectularius and lays the foundation for future functional genomics studies.

Quantitative real-time polymerase chain reaction (qRT-PCR) has emerged as robust methodology for gene expression studies, but reference genes are crucial for accurate normalization. Commonly used reference genes are housekeeping genes that are thought to be nonregulated; however, their expression can be unstable across different experimental conditions. We report the identification and validation of suitable reference genes in the bed bug, Cimex lectularius, by using qRT-PCR. The expression stability of eight reference genes in different tissues (abdominal cuticle, midgut, Malpighian tubules, and ovary) and developmental stages (early instar nymphs, late instar nymphs, and adults) of pesticide-susceptible and pesticide-exposed C. lectularius were analyzed using geNorm, NormFinder, and BestKeeper. Overall expression analysis of the eight reference genes revealed significant variation among samples, indicating the necessity of validating suitable reference genes for accurate quantification of mRNA transcripts. Ribosomal protein (RPL18) exhibited the most stable gene expression across all the tissue and developmental-stage samples; a-tubulin revealed the least stability across all of the samples examined. Thus, we recommend RPL18 as a suitable reference gene for normalization in gene expression studies of C. lectularius.

Summary Two hypotheses have been proposed to explain the origin of lifetime monogamy in the Isoptera. The classic explanation is that (1) the male must be present to continually provide sperm for the vast number of eggs produced by the queen (Snyder, 1924: Brian, 1983). Thornhill & Alcock (1983) proposed that (2) synchrony in the availability of receptive females necessitates mate guarding; males subsequently gain if they improve the relative reproductive success of their sole partner. Our review of the literature on termite flight behaviour, courtship behaviour, and incipient colony development indicates that neither of these two hypotheses satisfactorily explains the evolution of monogamy in termites. Because incipient colonies of lower termites exhibit a very low fecundity, it is doubtful that the continued presence of the male initially was due to the need for a continuous supply of spermatozoa. It is possible, however, that sperm requirements for the fertilization of numerous eggs over an extended period of time may be a factor in the persistence of the termites' monogamous mating system. Female alates are much more dispersed in time than implied by Thornhill & Alcock (1983) and there is no evidence of mate guarding. The importance of mate assistance is, however, supported by the literature. We propose a third hypothesis that incorporates the mate assistance element of the Thornhill & Alcock hypothesis: (3) the monogamous mating system of termites was structured by ecological constraints, namely, the low quality and scattered nature of their food/nesting material and the high costs of searching for a mate.

A census of 24 1-ha plots indicated .an average abundance per ha of 4.42 colonies of Reticulitermes flavipes (Kollar) and 2.38 colonies of R. virginicus (Banks). Nearest neighbor analysis indicated the mean distance between colonies of R. f2avipes to be 22.48 m, between colonies of R. virginicus to be 26.19 m, and between colonies, irrespective of species, to be 16.80 m. Six R. flavipes colonies were selected by a ranked-set sampling method, and the mean colony size was estimated to be 244,445 termites.

Blood-feeding insects have evolved resistance to various insecticides (organochlorines, pyrethroids, carbamates, etc.) through gene mutations and increased metabolism. Bed bugs (Cimex lectularius) are hematophagous ectoparasites that are poised to become one of the major pests in households throughout the United States. Currently, C. lectularius has attained a high global impact status due to its sudden and rampant resurgence. Resistance to pesticides is one factor implicated in this phenomenon. Although much emphasis has been placed on target sensitivity, little to no knowledge is available on the role of key metabolic players (e.g., cytochrome P450s and glutathione S-transferases) towards pesticide resistance in C. lectularius. In this review, we discuss different modes of resistance (target sensitivity, penetration resistance, behavioral resistance, and metabolic resistance) with more emphasis on metabolic resistance.

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Winter cover cropping plays an important role in conservation farming. In the Midwest region of the USA, strips planted with Austrian winter peas [AWP (Pisum sativum L.)], radishes [RAD (Raphanus sativus L.)], and a mixed cover of both cultivars (MIX) were compared during the spring season to determine their impact on the soil quality at 0-5 and 5-10 cm depths. The treatments RAD, AWP, and MIX had the highest, intermediate, and lowest soil bulk density impacts (1Á67, 1Á52, and 1Á50 Mg m À3 , respectively). An opposite trend was observed for the concentrations of soil organic carbon (15Á9, 17Á6, and 19Á4 g kg À1 , respectively) and total nitrogen (1Á5, 1Á7, and 2Á8 g kg À1 , respectively), the calcium exchange capacity (8Á6, 9Á5, and 11Á4 cmol + c kg À1 , respectively), and earthworm density (85, 221 and 226 per m 2 x 0Á15 m, respectively). The AWP, MIX, and RAD resulted in the highest, intermediate, and lowest water stable aggregates (91Á6, 87Á4, and 79Á9 per cent, respectively), mean weight diameter of aggregates (3Á4, 2Á7, and 2Á1 mm, respectively), saturated hydraulic conductivity (15Á7, 13Á0, and 6Á3 cm h À1 , respectively), and intrinsic permeability (1Á69, 1Á37, and 0Á70 cm 2 10 À10 , respectively). The AWP and RAD treatments resulted in a similar C:N ratio (10Á5), which was larger than associated with MIX (7Á2). Overall, AWP and MIX had a larger positive impact on soil quality than RAD did, and the impact was greater at the 0-5 than the 5-10 cm depth. This indicates the suitability of the various cover crops for improving the soil quality and restoring geodiversity.