Adaptation of Musca domestica L. Field Population to Laboratory Breeding Causes Transcriptional Alterations

Background

The housefly, Musca domestica, has developed resistance to most insecticides applied for its control. Expression of genes coding for detoxification enzymes play a role in the response of the housefly when encountered by a xenobiotic. The highest level of constitutive gene expression of nine P450 genes was previously found in a newly-collected susceptible field population in comparison to three insecticide-resistant laboratory strains and a laboratory reference strain.

Results

We compared gene expression of five P450s by qPCR as well as global gene expression by RNAseq in the newly-acquired field population (845b) in generation F₁, F₁₃ and F₂₉ to test how gene expression changes following laboratory adaption. Four (CYP6A1, CYP6A36, CYP6D3, CYP6G4) of five investigated P450 genes adapted to breeding by decreasing expression. CYP6D1 showed higher female expression in F₂₉ than in F₁. For males, about half of the genes accessed in the global gene expression were up-regulated in F₁₃ and F₂₉ in comparison with the F₁ population. In females, 60% of the genes were up-regulated in F₁₃ in comparison with F₁, while 33% were up-regulated in F₂₉. Forty potential P450 genes were identified. In most cases, P450 gene expression was decreased in F₁₃ flies in comparison with F₁. Gene expression then increased from F₁₃ to F₂₉ in males and decreased further in females.

Conclusion

The global gene expression changes massively during adaptation to laboratory breeding. In general, global expression decreased as a result of laboratory adaption in males, while female expression was not unidirectional. Expression of P450 genes was in general down-regulated as a result of laboratory adaption. Expression of hexamerin, coding for a storage protein was increased, while gene expression of genes coding for amylases decreased. This suggests a major impact of the surrounding environment on gene response to xenobiotics and genetic composition of housefly strains.     

Marker Assisted Transfer of Two Powdery Mildew Resistance Genes PmTb7A.1 and PmTb7A.2 from Triticum boeoticum (Boiss.) to Triticum aestivum (L.)

Powdery mildew (PM), caused by Blumeria graminis f. sp. tritici, is one of the important wheat diseases, worldwide. Two PM resistance genes, designated as PmTb7A.1 and PmTb7A.2, were identified in T. boeoticum acc. pau5088 and mapped on chromosome 7AL approximately 48cM apart. Two resistance gene analogue (RGA)-STS markers Ta7AL-4556232 and 7AL-4426363 were identified to be linked to the PmTb7A.1 and PmTb7A.2, at a distance of 0.6cM and 6.0cM, respectively. In the present study, following marker assisted selection (MAS), the two genes were transferred to T. aestivum using T. durum as bridging species. As many as 12,317 florets of F₁ of the cross T. durum /T. boeoticum were pollinated with T. aestivum lines PBW343-IL and PBW621 to produce 61 and 65 seeds, respectively, of three-way F₁. The resulting F₁s of the cross T. durum/T. boeoticum//T. aestivum were screened with marker flanking both the PM resistance genes PmTb7A.1 and PmTb7A.2 (foreground selection) and the selected plants were backcrossed to generate BC₁F₁. Marker assisted selection was carried both in BC₁F₁ and the BC₂F₁ generations. Introgression of alien chromatin in BC₂F₁ plants varied from 15.4 - 62.9 percent. Out of more than 110 BC₂F₁ plants showing introgression for markers linked to the two PM resistance genes, 40 agronomically desirable plants were selected for background selection for the carrier chromosome to identify the plants with minimum of the alien introgression. Cytological analysis showed that most plants have chromosome number ranging from 40-42. The BC₂F₂ plants homozygous for the two genes have been identified. These will be crossed to generate lines combining both the PM resistance genes but with minimal of the alien introgression. The PM resistance gene PmTb7A.1 maps in a region very close to Sr22, a stem rust resistance gene effective against the race Ug99. Analysis of selected plants with markers linked to Sr22 showed introgression of Sr22 from T. boeoticum in several BC₂F₁ plants. Thus, in addition to PM resistance, these progeny might also carry resistance to stem rust race Ug99.     

Functions of duplicated genes encoding CCAP receptors in the red flour beetle, Tribolium castaneum

Crustacean cardioactive peptide (CCAP) is a nonapeptide originally isolated from the shore crab, Carcinus maenas, based on its cardioacceleratory activity. This peptide is highly conserved in insects and other arthropods. In insects CCAP also has an essential role in ecdysis behavior. We previously identified two homologous genes, ccapr-1 and ccapr-2, encoding putative CCAP receptors in the red flour beetle, Tribolium castaneum. In contrast, some insects, including Drosophila melanogaster, carry only one gene encoding a CCAP receptor. Phylogenetic analysis of putative CCAP receptor orthologs reveals a number of independent gene duplications in several insect lineages. In this study, we confirmed that CCAP activates both putative T. castaneum receptors in a heterologous expression system. RNA interference (RNAi) of ccapr-1 and ccapr-2 revealed that ccapr-2 is essential for eclosion behavior in T. castaneum, while RNAi for ccapr-1 did not result in any abnormal phenotype. In vivo cardioacceleratory activity of exogenously applied CCAP was abolished by RNAi of ccapr-2, but not by that of ccapr-1. Thus, only ccapr-2 mediates the cardioacceleratory function, ccapr-1 having apparently lost both functions for eclosion behavior and for cardioacceleration since the recent gene duplication event.     

Characterization of the Critical Amino Acids of an Aspergillus parasiticus Cytochrome P-450 Monooxygenase Encoded by ordA That Is Involved in the Biosynthesis of Aflatoxins B1, G1, B2, and G2

The conversion of O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin to aflatoxins B₁, G₁, B₂, and G₂ requires a cytochrome P-450 type of oxidoreductase activity. ordA, a gene adjacent to the omtA gene, was identified in the aflatoxin-biosynthetic pathway gene cluster by chromosomal walking in Aspergillus parasiticus. The ordA gene was a homolog of the Aspergillus flavus ord1 gene, which is involved in the conversion of OMST to aflatoxin B₁. Complementation of A. parasiticus SRRC 2043, an OMST-accumulating strain, with the ordA gene restored the ability to produce aflatoxins B₁, G₁, B₂, and G₂. The ordA gene placed under the control of the GAL1 promoter converted exogenously supplied OMST to aflatoxin B₁ in Saccharomyces cerevisiae. In contrast, the ordA gene homolog in A. parasiticus SRRC 2043, ordA1, was not able to carry out the same conversion in the yeast system. Sequence analysis revealed that the ordA1 gene had three point mutations which resulted in three amino acid changes (His-400→Leu-400, Ala-143→Ser-143, and Ile-528→Tyr-528). Site-directed mutagenesis studies showed that the change of His-400 to Leu-400 resulted in a loss of the monooxygenase activity and that Ala-143 played a significant role in the catalytic conversion. In contrast, Ile-528 was not associated with the enzymatic activity. The involvement of the ordA gene in the synthesis of aflatoxins G₁, and G₂ in A. parasiticus suggests that enzymes required for the formation of aflatoxins G₁ and G₂ are not present in A. flavus. The results showed that in addition to the conserved heme-binding and redox reaction domains encoded by ordA, other seemingly domain-unrelated amino acid residues are critical for cytochrome P-450 catalytic activity. The ordA gene has been assigned to a new cytochrome P-450 gene family named CYP64 by The Cytochrome P450 Nomenclature Committee.     

Chromosomal location of genes for stem rust resistance derived from ‘Waldron’ wheat

The chromosomal locations of genes for resistance to stem rust (Puccinia graminis Pers.: Pers. f. sp. tritici Eriks. & E. Henn.) in the wheat (Triticum aestivum L.) cultivar ‘Waldron’ (WDR) were determined by monosomic analyses. Wheat lines WDR-B1, -C2, -E4, and -F1,which have single genes for resistance to stem rust derived previously from WDR sel. ‘Little Club’, were crossed onto a complete set of 21 ‘Chinese Spring’ monosomics. The F₂ and backcross-F₁ (BC₁F₁) seedlings from each of the 84 crosses were tested for reaction to culture 111-SS2 (CRL-LCBB) of stem rust, and a few selected segregants were analyzed cytologically for chromosome number. The F₂ from 2 crosses of WDR-C2, -E4 and -F1 and the BC₁F₁ from 2 crosses of WDR-F1 were tested also with culture Or11c (CRL-QBCN). Significant deviations from disomic ratios towards monosomic ratios in the F₂ and BC₁F₁ were used to determine which chromosomes carried the genes for resistance. Cytological analyses of certain BC₁F₁ and susceptible F₂ plants were used to help identify the location of the genes for rust resistance. WDR-B1 has a gene, herein designated Sr41, for resistance on chromosome 4D. WDR-C2 has a gene on chromosome 7 A that may be the same as one previously designated SrWld2. WDR-E4 has a gene on chromosome 2A, possibly SrWld1, which is effective against most or all North American stem rust cultures. WDR-F1 has a gene on chromosome 6B that is the same as or similar to Sr11.     

Normalization genes for mRNA expression in the marine diatom Ditylum brightwellii following exposure to thermal and toxic chemical stresses

Toxicity assessments using the diatom Ditylum brightwellii are well documented; however, analysis of their toxicogenomics has been little attempted. Currently, quantitative real-time PCR is the most accurate and widely applied method to detect differential gene expression, including that of specific genes induced by environmental contaminants. This method requires internal reference genes to normalize expression levels, and their selection is a critical factor for the correct analysis of the results. Here, we assessed the gene expression stability of nine housekeeping genes (HKGs), including 18S rRNA, ACT, TUA, EF2, MDH, UBQ, UCE, PCNA, and GAPDH, in 28 RNA samples of D. brightwellii. All the tested HKGs displayed different expression patterns under different experimental conditions such as heat shock and exposure to metals and non-metals. Analysis of C _T values showed that at least two genes were required for proper normalization according to the tested conditions. Overall, TUA, followed by ACT, was the most stable gene under all conditions. Furthermore, we examined the expression of the HSP70 gene in D. brightwellii when exposed to heat shock and chemicals by using the most stable references and found that the gene was significantly up-regulated during the stress period. This study has evaluated, for the first time, the normalization genes in D. brightwellii, providing potential references for gene expression studies of diatoms.     

The effects of long-term estradiol treatment on social behavior and gene expression in adult female rats

This study tested the effects of long-term estradiol (E₂) replacement on social behavior and gene expression in brain nuclei involved in the regulation of these social behaviors in adult female rats. We developed an ultrasonic vocalization (USV) test and a sociability test to examine communications, social interactions, and social preference, using young adult female cagemates. All rats were ovariectomized (OVX) and implanted with a Silastic capsule containing E₂ or vehicle, and housed in same-treatment pairs for a 3-month period. Then, rats were behaviorally tested, euthanized, and 5 nuclei in the brain's social decision-making circuit were selected for neuromolecular profiling by a multiplex qPCR method. Our novel USV test proved to be a robust tool to measure numbers and types of calls emitted by cagemates that had been reintroduced after a 1-week separation. Results also showed that E₂-treated OVX rats had profoundly decreased numbers of USV calls compared to vehicle-treated OVX rats. In a test of sociability, in which a female was allowed to choose between her cagemate or a same-treatment novel rat, we found few effects of E₂ compared to vehicle, although interestingly, rats chose the cagemate over an unfamiliar conspecific. Gene expression results revealed that the supraoptic nucleus had the greatest number of gene changes caused by E₂: Oxt, Oxtr and Avp were increased, and Drd2, Htr1a, Grin2b, and Gabbr1 were decreased, by E₂. No genes were affected in the prefrontal cortex, and 1–4 genes were changed in paraventricular nucleus (Pgr), bed nucleus of the stria terminalis (Oxtr, Esr2, Dnmt3a), and medial amygdala (Oxtr, Ar, Foxp1, Tac3). Thus, E₂ changes communicative interactions between adult female rats, together with selected expression of genes in the brain, especially in the supraoptic nucleus.