Behavioral responses and life history traits of Taiwanese and Indonesian populations of Aedes aegypti surviving deltamethrin-clothianidin treatment.

BACKGROUND: Indoor residual spraying (IRS) capitalizes on the natural behavior of mosquitoes because Aedes aegypti commonly seeks indoor resting sites after a blood meal. This behavior allows mosquitoes to be exposed to insecticide-treated surfaces and subsequently killed. Combinations of deltamethrin and clothianidin with different modes of action have shown promise in IRS, effectively targeting both susceptible and pyrethroid-resistant malaria vectors. However, the effects of this approach on Aedes mosquitoes remain unclear. The present study tested the effects of deltamethrin-clothianidin mixture treatment on behavioral responses and life history traits of Taiwanese and Indonesian populations of Ae. aegypti. METHODS: We adopted an excito-repellent approach to explore the behavioral responses of pyrethroid-resistant Ae. aegypti populations from Indonesia and Taiwan to a deltamethrin-clothianidin mixture used in contact irritancy and non-contact repellency treatments. We further evaluated the life history traits of surviving mosquitoes (i.e., delayed mortality after 7-day post-treatment, longevity, fecundity, and egg hatching) and investigated the potential transgenerational hormetic effects of insecticide exposure (i.e., development rate and survival of immatures and adult mosquitos). RESULTS: All tested field populations of Ae. aegypti displayed strong contact irritancy responses; the percentage of escape upon insecticide exposure ranged from 38.8% to 84.7%. However, repellent effects were limited, with the escape percentage ranging from 4.3% to 48.9%. We did not observe immediate knockdown or mortality after 24 h, and less than 15% of the mosquitoes exhibited delayed mortality after a 7-day exposure period. However, the carryover effects of insecticide exposure on the survival of immature mosquitoes resulted in approximately 25% higher immature mortality than that in the control. By contrast, we further documented stimulated survivor reproduction and accelerated transgenerational immature development resulting from the sublethal effects of the insecticide mixture. In particular, the number of eggs laid by treated (both treatments) female mosquitoes increased by at least 60% compared with that of eggs laid by control female mosquitoes. CONCLUSIONS: IRS with deltamethrin-clothianidin effectively deters Aedes mosquitoes from entering residential areas and thereby reduces mosquito bites. However, the application rate (deltamethrin: 25 mg/m2; clothianidin: 200 mg/m2) may be insufficient to effectively kill Aedes mosquitoes. Insecticide response appears to vary across mosquito species; their behavioral and physiological responses to sublethal doses have crucial implications for mosquito control programs.

Regulation of Aquaporin Prip Expression and Its Physiological Function in Rhyzopertha dominica (Coleoptera: Bostrichidae).

Rhyzopertha dominica Prip (RdPrip) cDNA was cloned (GenBank accession no. OK318454), and the encoded 276-amino-acid protein indicated the typical aquaporin structure, including six transmembrane regions and two NPA motifs. The developmental and tissue profiles of RdPrip transcription were determined. RdPrip was highly transcribed in female adults, followed by larvae, pupae, and male adults. The transcriptional expression levels of RdPrip were significantly high in the ovary and hindgut (including cryptonephridial systems) compared with the foregut, testis, midgut, and Malpighian tubules. Knockdown of RdPrip in female adults did not decrease fecundity, but significantly decreased the hatching rate of eggs laid by the females. The results suggest that RdPrip functions in embryonic development, not in egg formation. In addition, the transcriptional expression level of RdPrip was lower in the spinosad-resistant strain than in the susceptible one, and the resistant strain produced fewer progeny than the susceptible strain did. These studies support the functional role of RdPrip in female reproduction. The absence of significant mortality reduction in the R. dominica exposed to spinosad after RdPrip RNAi suggests that other aquaporins that were not knocked down may exist for the excretion of metabolized pesticides.

Association of CYP4G19 Expression With Gel Bait Performance in Pyrethroid-Resistant German Cockroaches (Blattodea: Ectobiidae) From Taiwan.

Overexpression of a cytochrome P450 gene, CYP4G19, is known to associate with pyrethroid resistance in the German cockroach, Blattella germanica (L.) (Blattodea: Ectobiidae). In this study, we investigated the CYP4G19 expression level in 20 field-collected strains of B. germanica in Taiwan. We also examined the level of adult male susceptibility to imidacloprid, fipronil, indoxacarb, and hydramethylnon using single-diagnostic dose bioassays and their susceptibility to corresponding gel baits to determine how the CYP4G19 expression level influences the cockroach gel bait performance. Results showed that the CYP4G19 gene expression level among the field-collected German cockroach was 1.8- to 9.7-fold higher than that of the susceptible strain. It was negatively correlated (P < 0.05) with the % mortality after treatments with imidacloprid and fipronil diagnostic doses. However, no correlation was found between CYP4G19 gene expression with the % mortality after treatment with indoxacarb and hydramethylnon diagnostic doses. Indoxacarb and hydramethylnon baits showed high efficacy against the field strains with a mean mortality of 97.58 ± 1.35% and 90.95 ±1.65%, respectively. This study provided the first evidence of cross-resistance to imidacloprid and fipronil in pyrethroid-resistant German cockroaches due to overexpression of CYP4G19.

Molecular Characteristics of Fat Body Protein 1 in the Oriental Fruit Fly, Bactrocera dorsalis.

Bactrocera dorsails fat body protein 1 (Bdfbp1) cDNA was cloned (GenBank accession no. MT514270), and the complete 3,749-bp cDNA encoded a 1,152-amino acid protein. The phylogenetic relationship of dipteran fbp1s was analyzed. The sequence XP_028900815 from the insect genome project for Zeugodacus cucurbitae (LOC105219342) was proposed that two fbp1 genes were present in the sequence. The developmental transcriptional expression profiles were determined. In the larval stages, Bdfbp1 mRNA had significantly higher expression in the late third instar larvae compared with first, second, and early third instar larvae. In the pupal stages, the highest expression of Bdfbp1 mRNA was found in the newly pupated pupae and then decreased with age. In the fat body of female adults, Bdfbp1 was highly expressed in newly emerged samples and decreased rapidly over the following three days. In the fat body of male adults, Bdfbp1 was highly expressed in newly eclosed samples. RNAi treatment decreased the expression level of Bdfbp1 without statistical difference. However, RNAi treatment significantly decreased the rate of eclosion. These results suggest that Bdfbp1 may function as a storage protein and be associated with adult eclosion.

Nicotinic acetylcholine receptor subunit α6 associated with spinosad resistance in Rhyzopertha dominica (Coleoptera: Bostrichidae).

The lesser grain borer, Rhyzopertha dominica, which is a primary pest of stored products, breaks up whole grains and makes them susceptible to secondary infestation by other pests. Insecticide application is the main control measure against this borer. A resistant strain of R. dominica against the insecticide, spinosad, was selected in the laboratory. The full-length cDNA of the target site of spinosad, nicotinic acetylcholine receptor subunit α6, from R. dominica (Rdα6) was cloned and analyzed using reverse transcription PCR and rapid amplification of cDNA ends. The complete 2133-bp cDNA contains the open reading frame of 1497 bp encoding a 498-amino-acid protein. There are four predicted transmembrane (TM) regions, and six extracellular ligand-binding sites at the N-terminus, upstream from the first TM in Rdα6. Three mutations have been found in the resistant strain compared with the susceptible one: (1) a 181-bp fragment truncated at the N-terminus, resulting in the appearance of a premature stop codon, (2) one missing bp at the position 997, causing a frame-shift mutation, and (3) an 87-bp fragment truncated in the TM2 region. In addition, real-time quantitative PCR was applied to detect the transcriptional expression of Rdα6 in both the susceptible and resistant strains. The results indicated that the expression of Rdα6 was significantly lower in then resistant strain than in susceptible one. In conclusion, mutation of Rdα6 may cause R. dominica resistant to spinosad due to target site insensitivity.

Effects of glycation on human γd-crystallin proteins by different glycation-inducing agents.

Human γd-crystallin (Hγd-crystallin), a major protein component of the human eye lens, is associated with the development of juvenile- and mature-onset cataracts. Evidence suggests that nonenzymatic protein glycation plays an important role in the aetiology of cataract and diabetic sequelae. This research compared the effects of various glycation modifiers on Hγd-crystallin aggregation, by treating samples of Hγd-crystallin with ribose, galactose, or methylglyoxal using several biophysical techniques. To measure advanced glycation end products, an Nε-(carboxyethyl)lysine enzyme-linked immunosorbent assay was performed on the glycating agent-treated Hγd-crystallin samples. Fructosamine production detection was performed for both ribose-treated and galactose-treated samples. Methylglyoxal-treated samples had the highest level of aggregation and the greatest extent of unfolding, and upon incubation for a minimum of 12 days, exhibited a marked enhancement in the amount of Nε-(carboxyethyl)lysine. The molecular profiles and morphological features of the glycated samples were highly correlated to the type of glycation agent used. These findings highlight a close connection between the type of glycation modifier and the various aggregation species that form. Thus, these results may facilitate deciphering of the molecular mechanism of diabetic cataractogenesis.

Brain gene expression analyses in virgin and mated queens of fire ants reveal mating-independent and socially regulated changes.

Transcriptomes of dissected brains from virgin alate and dealate mated queens from polygyne fire ants (Solenopsis invicta) were analyzed and compared. Thirteen genes were upregulated in mated queen brain, and nine were downregulated. While many of the regulated genes were either uncharacterized or noncoding RNAs, those annotated genes included two hexamerin proteins, astakine neuropeptide, serine proteases, and serine protease inhibitors. We found that for select differentially expressed genes in the brain, changes in gene expression were most likely driven by the changes in physiological state (i.e., age, nutritional status, or dominance rank) or in social environment (released from influence of primer pheromone). This was concluded because virgins that dealated after being separated from mated queens showed similar patterns of gene expression in the brain as those of mated queens for hexamerin 1, astakine, and XR_850909. Abaecin (XR_850725), however, appears upregulated only after mating. Therefore, our findings contribute to distinguish how specific gene networks, especially those influenced by queen primer pheromone, are regulated in queen ants. Additionally, to identify brain signaling pathways, we mined the fire ant genome and compiled a list of G-protein-coupled receptors (GPCRs). The expression level of GPCRs and other genes in the "genetic toolkit" in the brains of virgin alates and mated dealate queens is reported.

Complete mitochondrial genome of Aeolesthes oenochrous (Fairmaire) (Coleoptera: Cerambycidae): an endangered and colorful longhorn beetle.

Aeolesthes oenochrous (Fairmaire), a large and colorful longhorn beetle, is an endangered species in Taiwan. Its complete mitogenome, 15,747 bp, shows a typical coleopteran organization, containing 13 protein coding genes, 22 tRNA genes, 2 rRNA genes and one A + T rich region. Two protein coding genes, i.e. COI and ND1, have the atypical start codon of AAT and TTG, respectively. The third nucleotide position of codons shows extremely low guanine content. In the A + T rich region, there were two poly-T stretches with 14 and 13 thymine each. These two poly-T stretches were clarified by the cloning method.

A NONOVARY-SPECIFIC VITELLOGENIN RECEPTOR FROM THE ORIENTAL FRUIT FLY, Bactrocera dorsalis (HENDEL).

The yolk protein precursor, vitellogenin (Vg), is absorbed into growing oocytes via receptor-mediated endocytosis for embryonic development. In this study, a Vg receptor (VgR) cDNA of the oriental fruit fly (Bactrocera dorsalis Hendel) was cloned via RT-PCR and RACE (GenBank accession no. KR535603) and its expression analyzed. The BdVgR cDNA has a length of 6,585 bp encoding 1,923 amino acids. It has a conserved motif arrangement with other insect VgRs, and showed high identity to the B. cucurbitae VgR (91.4%). The expression of BdVgR mRNA and proteins was shown in both ovary and fat body. This is the first report on a nonovary-specific VgR from a nonsocial insect. In ovary, the expression of BdVgR mRNA and proteins was inconsistent, with the transcription, but not protein, level high on D0. In fat body, the expression levels of BdVgR mRNA and proteins were high on days 5 and 6. The function of BdVgR in the fat body is not clear. However, it may be involved in reuptake of yolk proteins from the hemolymph as an amino acid reservoir or as autocrine regulation of yolk protein expression.

cDNA CLONING AND TRANSCRIPTIONAL REGULATION OF THE CECROPIN AND ATTACIN FROM THE ORIENTAL FRUIT FLY, Bactrocera dorsalis (DIPTERA: TEPHRITIDAE).

We described the cDNA cloning of two antimicrobial peptides (AMPs), cecropin (BdCec), and attacin C (BdAttC), from the oriental fruit fly, Bactrocera dorsalis (Hendel), a serious insect pest of fruit trees. Using rapid amplification of cDNA ends, fragments encompassing the entire open reading frames of BdCec and BdAttC were cloned and sequenced. The complete 425 bp cDNA of BdCec encodes a protein of 64 amino acids with a predicted molecular weight of 6.84 kDa. The 931 bp cDNA of BdAttC encodes a protein of 239 residues with a predicted molecular weight of 24.97 kDa. Real-time quantitative RT-PCR demonstrated that the developmental transcription profiles of BdCec and BdAttC were similar in each larvae, pupae, and adults. The constitutive expression levels of both AMPs were high in the first-instar and late third-instar larvae, suggesting that their antimicrobial activity is active in the newly hatched larvae and just before pupation. The basal expression levels were not significant different in adult fat bodies. The expression of BdCec and BdAttC was upregulated after bacterial challenge in adult fat bodies. The ratio of inducible expression to constitutive expression was lower in males compared to females.

Comparative analysis of human γD-crystallin aggregation under physiological and low pH conditions.

Cataract, a major cause of visual impairment worldwide, is the opacification of the eye's crystalline lens due to aggregation of the crystallin proteins. The research reported here is aimed at investigating the aggregating behavior of γ-crystallin proteins in various incubation conditions. Thioflavin T binding assay, circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, intrinsic (tryptophan) fluorescence spectroscopy, light scattering, and electron microscopy were used for structural characterization. Molecular dynamics simulations and bioinformatics prediction were performed to gain insights into the γD-crystallin mechanisms of fibrillogenesis. We first demonstrated that, except at pH 7.0 and 37°C, the aggregation of γD-crystallin was observed to be augmented upon incubation, as revealed by turbidity measurements. Next, the types of aggregates (fibrillar or non-fibrillar aggregates) formed under different incubation conditions were identified. We found that, while a variety of non-fibrillar, granular species were detected in the sample incubated under pH 7.0, the fibrillogenesis of human γD-crystallin could be induced by acidic pH (pH 2.0). In addition, circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, and intrinsic fluorescence spectroscopy were used to characterize the structural and conformational features in different incubation conditions. Our results suggested that incubation under acidic condition led to a considerable change in the secondary structure and an enhancement in solvent-exposure of the hydrophobic regions of human γD-crystallin. Finally, molecular dynamics simulations and bioinformatics prediction were performed to better explain the differences between the structures and/or conformations of the human γD-crystallin samples and to reveal potential key protein region involved in the varied aggregation behavior. Bioinformatics analyses revealed that the initiation of amyloid formation of human γD-crystallin may be associated with a region within the C-terminal domain. We believe the results from this research may contribute to a better understanding of the possible mechanisms underlying the pathogenesis of senile nuclear cataract.

cDNA cloning and transcriptional expression profiles of a hexamerin in the oriental fruit fly, Bactrocera dorsalis.

A Bactrocera dorsalis hexamerin (BdAr) cDNA was cloned (GenBank accession no. KF815528), and its transcriptional expression profiles were determined. The complete 2,530-bp cDNA encodes a 780-amino acid protein with a predicted molecular mass of 94.01 kDa. The proportions of phenylalanine (7.8%), tyrosine (11.2%), and methionine (2.6%) in BdAr as well as all other amino acids are reported. BdAr transcripts were detected in the brain, flight muscle, foregut, Malpighian tubules, and fat body. In the larval stage, BdAr transcripts were expressed in the early third instar and increased in the late third instar. In pupae, the highest expression of BdAr mRNA was present on day 1, then declined and persisted through day 2 to day 8. In adult females, the relative expression of BdAr was significantly higher on day 0 and day 1 compared to day 6 to day 10 while it was highest in newly eclosed adult males. The comparison of the BdAr expression between 8-10-day-old males and females showed a higher level in females. Our phylogenetic analysis results suggest to us that BdAr is similar to Drosophila larval serum protein 1γ.

Differential gene expression in male and female fat body in the oriental fruit fly, Bactrocera dorsalis.

The sexual difference in gene expression in fat body between 8- and 10-day-old male and female Bactrocera dorsalis was examined using suppression subtractive hybridization. A total of 952 clones were sequenced and searched using BLAST from the subtracted cDNA library. About 22% of these clones showed homology with detoxification enzymes including cytochrome P450 monooxygenases (CYPs) and glutathione S-transferase. NADH dehydrogenases, distributed to energy metabolism, constituted about 9% of these clones. About 10% of these clones were cecropin, an antimicrobial peptide. Real-time quantitative polymerase chain reaction (qPCR) analysis showed that four transcripts were expressed at a higher level in fat body of males, compared to females. Bactrocera dorsalis cyp6g2 (Bdcyp6g2) was cloned (accession number KF469179) and the temporal profile of transcriptional expression showed that Bdcyp6g2 mRNA increased with age in males from day 3 after eclosion, but only on days 0-3 in females. Compared to females, the susceptibility of 9-day-old males to three insecticides was significantly less. These results suggested the genes expressed at a higher level in male act in its survival.

Glucose transporter 8 (GLUT8) from the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae).

We have cloned the fire ant glucose transporter 8 (GLUT8) cDNA providing the first molecular characterization of a GLUT8 in insects. Glucose is a poly-alcohol and, due to its high hydrophilicity, cannot move across cell membranes. GLUT8 is a putative facilitative transporter for the cellular import and export of glucose. The complete 2,974-bp cDNA encodes a 501-residue protein with a predicted molecular mass of 54.8 kDa. Transcripts were detected in the brain, midgut, hindgut, Malpighian tubule, fat body, ovary, and testis. The highest transcriptional expression was found in fat body. Northern blot analysis revealed different transcript sizes in mated queen brains, alate female ovaries, and male testes. We propose that four other sequences obtained from insect genome projects from the honey bee Apis mellifera (ENSAPMP00000006624), the malaria mosquito Anopheles gambiae (EAA11842), and the fruit fly Drosophila melanogaster (AAQ23604 and AAM52591) are likely the orthologues of the fire ant GLUT8. Phylogenetic relationships in insect glucose transporters are presented.

The short neuropeptide F-like receptor from the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae).

In invertebrates, neuropeptide F (NPF) peptides share structural similarity with vertebrate neuropeptide Y, which regulates food consumption, circadian rhythms, anxiety, and other physiological processes. The insect neuropeptide F receptors belong to the G protein-coupled receptor (GPCR) rhodopsin family. We have cloned the fire ant putative short NPF receptor using PCR and RACE methods. The complete 2,185-bp cDNA encodes a 387-residue protein with a predicted GPCR seven transmembrane region structure. We propose that the sequence of the honey bee short NPF receptor, which has not yet been annotated, encodes a protein of 393 residues. In fire ant mated queens, receptor transcripts were detected in the brain, midgut, hindgut, Malpighian tubules, fat body, and ovaries. The highest transcriptional expression was found in the brain. The downregulation of the fire ant short NPF receptor transcriptional expression in the brain with starvation suggests that the short NPF signal transduction cascade may play a role in feeding regulation in fire ant mated queens.