Advancing quantitative PCR with color cycle multiplex amplification.

Quantitative PCR (qPCR) is the gold standard for detection and quantitation of known DNA targets, but the scarcity of spectrally distinct fluorophores and filter sets limits the number of detectable targets. Here, we introduce color cycle multiplex amplification (CCMA) to significantly increase the number of detectable DNA targets in a single qPCR reaction using standard instrumentation. In CCMA, presence of one DNA target species results in a pre-programmed pattern of fluorescence increases. This pattern is distinguished by cycle thresholds (Cts) through rationally designed delays in amplification. For example, we design an assay wherein Staphylococcus aureus sequentially induces FAM, then Cy5.5, then ROX fluorescence increases with more than 3 cycles between each signal. CCMA offers notably higher potential for multiplexing because it uses fluorescence permutation rather than combination. With 4 distinct fluorescence colors, CCMA theoretically allows the detection of up to 136 distinct DNA target sequences using fluorescence permutation. Experimentally, we demonstrated a single-tube qPCR assay screening 21 sepsis-related bacterial DNA targets in samples of blood, sputum, pleural effusion and bronchoalveolar lavage fluid, with 89% clinical sensitivity and 100% clinical specificity, showing its potential as a powerful tool for advanced quantitative screening in molecular diagnostics.

Systematic revision of the North American velvet ant genus Invreiella Suárez (Hymenoptera: Mutillidae) with description of eleven new species.

The velvet ant genus Invreiella Suárez, 1966 is redescribed and revised. Females are grouped into five species-groups, with eleven new species described based on females: I. acuminata Waldren, sp. nov., I. australis Waldren, sp. nov., I. bimaculata Waldren, sp. nov., I. breviclypeata Waldren, sp. nov., I. chihuahuensis Waldren, sp. nov., I. cuernavaca Waldren, sp. nov., I. erythrocephala Waldren, sp. nov., I. manleyi Waldren, sp. nov., I. mesomexicana Waldren, sp. nov., I. suarezi Waldren, sp. nov., and I. tequila Waldren, sp. nov. One new combination is included, I. cephalargia (Mickel, 1924), comb. nov., formerly placed in Pseudomethoca Ashmead, 1896. Additionally, I. curoei Quintero Cambra, 2011 is synonymized with I. cardinalis (Gerstaecker, 1874), syn. nov., and I. megacantha (Cockerell Casad, 1894), stat. resurr., is treated as a nomen dubium. The lectotype of Mutilla satrapa Gerstaecker, 1874 originally designated by C. Mickel is here validated. An illustrated key to species is included.

Observation of a mixed-sex, mixed-species aggregation of velvet ants, genus Timulla Ashmead, 1899 (Hymenoptera: Mutillidae) in the Brazilian Amazon, Roraima, with a new synonymy.

An aggregation of two males of Timulla bitaeniata (Spinola, 1841), three single males T. rufogastra (Lepeletier, 1845) and another two males T. rufogastra (Lepeletier, 1845) in copula with two females of T. eriphyla Mickel, 1938 is documented, described and discussed. A new synonymy is proposed for Mutilla rufogastra Lepeletier de Saint-Fargeau, 1845 [=Timulla (Timulla) eriphyla Mickel, 1938, syn. nov.] based on the mating pairs.

Bees as hosts of mutillid wasps in the Neotropical region (Hymenoptera, Apidae, Mutillidae)

ABSTRACT A review of bee species used as hosts of mutillid wasps in the Neotropical region is presented. Three new confirmed host records are provided for the mutillid species Hoplomutilla biplagiata Mickel, 1939, Pappognatha limes Mickel, 1939, and Tallium aracati Casal, 1962. Two potential host records are provided for Euspinolia rufula Mickel, 1938 and Lophomutilla inca Fritz and Pagliano, 1993. Additionally, Mutilla hoplitiformis Strand, 1909, is transferred to the genus Darditilla. Correlations between host nesting habits and female mutillid morphology are discussed. Lastly, all known confirmed and potential host records in the Neotropical region are compiled.