Functional synthetic Antennapedia genes and the dual roles of YPWM motif and linker size in transcriptional activation and repression.

Segmental identity along the anteroposterior axis of bilateral animals is specified by Hox genes. These genes encode transcription factors, harboring the conserved homeodomain and, generally, a YPWM motif, which binds Hox cofactors and increases Hox transcriptional specificity in vivo. Here we derive synthetic Drosophila Antennapedia genes, consisting only of the YPWM motif and homeodomain, and investigate their functional role throughout development. Synthetic peptides and full-length Antennapedia proteins cause head-to-thorax transformations in the embryo, as well as antenna-to-tarsus and eye-to-wing transformations in the adult, thus converting the entire head to a mesothorax. This conversion is achieved by repression of genes required for head and antennal development and ectopic activation of genes promoting thoracic and tarsal fates, respectively. Synthetic Antennapedia peptides bind DNA specifically and interact with Extradenticle and Bric-à-brac interacting protein 2 cofactors in vitro and ex vivo. Substitution of the YPWM motif by alanines abolishes Antennapedia homeotic function, whereas substitution of YPWM by the WRPW repressor motif, which binds the transcriptional corepressor Groucho, allows all proteins to act as repressors only. Finally, naturally occurring variations in the size of the linker between the homeodomain and YPWM motif enhance Antennapedia repressive or activating efficiency, emphasizing the importance of linker size, rather than sequence, for specificity. Our results clearly show that synthetic Antennapedia genes are functional in vivo and therefore provide powerful tools for synthetic biology. Moreover, the YPWM motif is necessary--whereas the entire N terminus of the protein is dispensable--for Antennapedia homeotic function, indicating its dual role in transcriptional activation and repression by recruiting either coactivators or corepressors.

Impact of deltamethrin selection on kdr mutations and insecticide detoxifying enzymes in Aedes aegypti from Mexico.

BACKGROUND: Insecticide resistance is a serious problem for vector control programmes worldwide. Resistance is commonly attributed to mutations at the insecticide's target site or increased activity of detoxification enzymes. METHODS: We determined the knockdown concentration (KC50) and lethal concentration (LC50) of deltamethrin in six natural populations of adult Aedes aegypti from southeastern Mexico. These populations were then selected over five generations using the LC50 from the preceding generation that underwent selection, and the heritability of deltamethrin resistance was quantified. For each generation, we also determined the frequency of the kdr alleles L410, I1016 and C1534, and the levels of activity of three enzyme families (α- and ß-esterases, mixed-function oxidases and glutathione S-transferases (GST)) associated with insecticide detoxification. RESULTS: There was an increase in KC50 and LC50 values in the subsequent generations of selection with deltamethrin (FS5vs FS0). According to the resistance ratios (RRs), we detected increases in LC50 ranging from 1.5 to 5.6 times the values of the parental generation and in KC50 ranging from 1.3-3.8 times the values of the parental generation. Triple homozygous mutant individuals (tri-locus, LL/II/CC) were present in the parental generations and increased in frequency after selection. The frequency of L410 increased from 1.18-fold to 2.63-fold after selection with deltamethrin (FS5vs FS0) in the populations analyzed; for I1016 an increase between 1.19-fold to 2.79-fold was observed, and C1534 was fixed in all populations after deltamethrin selection. Enzymatic activity varied significantly over the generations of selection. However, only α- esterase activity remained elevated in multiple populations after five generations of deltamethrin selection. We observed an increase in the mean activity levels of GSTs in two of the six populations analyzed. CONCLUSIONS: The high levels of resistance and their association with high frequencies of kdr mutations (V410L, V1016I and F1534C) obtained through artificial selection, suggest an important role of these mutations in conferring resistance to deltamethrin. We highlight the need to implement strategies that involve the monitoring of kdr frequencies in insecticide resistance monitoring and management programmes.

Multiplex PCR for simultaneous genotyping of kdr mutations V410L, V1016I and F1534C in Aedes aegypti (L.).

BACKGROUND: Knockdown resistance (kdr) is the main mechanism that confers resistance to pyrethroids and DDT. This is a product of non-synonymous mutations in the voltage-gated sodium channel (vgsc) gene, and these mutations produce a change of a single amino acid which reduces the affinity of the target site for the insecticide molecule. In Mexico, V410L, V1016I and F1534C mutations are common in pyrethroid-resistant Aedes aegypti (L.) populations. METHODS: A multiplex PCR was developed to detect the V410L, V1016I and F1534C mutations in Ae. aegypti. The validation of the technique was carried out by DNA sequencing using field populations previously characterized for the three mutations through allele-specific PCR (AS-PCR) and with different levels of genotypic frequencies. RESULTS: The standardized protocol for multiplex end-point PCR was highly effective in detecting 15 genotypes considering the three mutations V410L, V1106I and F1534C, in 12 field populations of Ae. aegypti from Mexico. A complete concordance with AS-PCR and DNA sequencing was found for the simultaneous detection of the three kdr mutations. CONCLUSIONS: Our diagnostic method is highly effective for the simultaneous detection of V410L, V1016I and F1534C, when they co-occur. This technique represents a viable alternative to complement and strengthen current monitoring and resistance management strategies against Ae. aegypti.

Report of Chikungunya Virus in Wild Populations of Aedes aegypti in Guerrero State, Mexico.

We detected vertical transmission of chikungunya virus (CHIKV) in wild populations of Aedes aegypti from San Marcos, Guerrero, Mexico, with real-time reverse transcriptase-polymerase chain reaction. A total of 20 pools (1-11 specimens/pool) of larvae, male, and female mosquitoes were tested. We report the detection of CHIKV in 2 of 11 larval pools, 4 of 5 male pools, and 1 of 4 female pools, from field-collected mosquitoes.

First Detection of the Kdr Mutation T929I in Head Lice (Phthiraptera: Pediculidae) in Schoolchildren of the Metropolitan Area of Nuevo Leon and Yucatan, Mexico.

The head louse Pediculus humanus capitis (De Geer) is a hematophagous ectoparasite that inhabits the human scalp. Infestations by this insect are commonly known as pediculosis, which is more common in younger groups. These infestations are asymptomatic; however, skin irritation from scratching occasionally may cause secondary bacterial infections. In recent years, the prevalence of pediculosis has increased in children; this increase has been attributed to louse resistance to the insecticides used as a control measure for infestation. The aim of the present study was to determine the presence and frequency of the knockdown resistance mutation (kdr) T929I in 468 head lice collected from 32 elementary schools in the metropolitan area of Nuevo Leon (24) and Yucatan (8), Mexico. This is the first report of a knockdown resistance (kdr) mechanism in head lice from Mexico. The T929I mutation was present in all of the sampled schools, with variability observed in its allelic and genotypic frequencies.

Knockdown Resistance Mutations in Aedes aegypti (Diptera: Culicidae) From Puerto Rico.

Permethrin resistance is widespread in Aedes aegypti (L.), the main dengue, zika, and chikungunya virus vector in Latin America and the Caribbean. A common mechanism of resistance to pyrethroids-knockdown resistance (kdr)-is conferred through mutations in the insect's voltage-dependent sodium channel. In this mosquito, around 10 replacement substitutions in the voltage-gated sodium channel gene (vgsc) have been reported in pyrethroid-resistant strains. Two of these mutations, named Ile1,016 and Cys1,534, are widespread in mosquito populations from Latin America and the Caribbean. This study assessed the levels of permethrin resistance and the frequency of two kdr mutations in eight Ae. aegypti populations collected in Puerto Rico in 2013. Permethrin resistance factors ranged from 33-214-fold relative to the New Orleans reference strain. The frequency of kdr mutation Ile1,016 ranged from 0.65 to fixation (1.0), and for Cys1,534 frequencies varied from 0.8 to fixation. Alarmingly, two populations-Carolina and Caguas-reached fixation at both loci. Our results suggest that permethrin effectiveness for Ae. aegypti control is compromised in these collections from Puerto Rico.

Novel Kdr mutations (K964R and A943V) in pyrethroid-resistant populations of Triatoma mazzottii and Triatoma longipennis from Mexico and detoxifying enzymes

Although having five different ways of transmission the vector-borne is the principal way of transmission of Chagas disease, which involves insects of the subfamily Triatominae (Hemiptera: Reduviidae). Nineteen of the 31 species that occur in Mexico are associated with humans, and all are capable of transmitting the disease. Pyrethroids are the insecticides recommended for the control of these vectors in Mexico. We determined the susceptibility to the pyrethroids deltamethrin and permethrin of peridomestic populations of Triatoma mazzottii Usinger and two populations of Triatoma longipennis Usinger in comparison with a reference strain for each species. Bioassays were performed for the determination of the LD50 for both field populations and reference strains. A maximum of 27 fold resistance to deltamethrin was observed in T. mazzottii, meanwhile, for permethrin, T. longipennis from Jalisco show the highest value of 3.19 fold. There was significantly increased activity of esterases in field populations in comparison with their corresponding reference strain. The results of the search of kdr mutations related to the resistance to deltamethrin and permethrin in the evaluated species show the presence of mutations in the field populations, as is the case with individuals of T. mazzottii, for which the mutation was found A943V, and for the two populations of T. longipennis included in this study, we report the presence of the kdr mutation K964R. Evaluation of the various mechanisms involved in resistance to pyrethroids in triatomines from Mexico could guide us to the real justification for insecticide resistance monitoring.