Abstract
The German cockroach, Blattella germanica, and the American cockroach, Periplaneta americana are the most common and synanthropic household pests of interest to public health. While they have increasingly served as model systems in hemimetabolous insects for studying many biological issues, there is still a lack of stable reference gene evaluation for reliable quantitative real-time PCR (qPCR) outputs and functional genomics. Here, we evaluated the expression variation of common insect reference genes, including the historically used actin, across various tissues and developmental stages, and also under experimental treatment conditions in these two species by using three individual algorithms (geNorm, BestKeeper, and NormFinder) and a comprehensive program (RefFinder). RPL32 in B. germanica and EF1α in P. americana showed the overall lowest variation among all examined samples. Based on the stability rankings by RefFinder, the optimal but varied reference genes under specific conditions were selected for qPCR normalization. In addition, the combination of RPL32 and EF1α was recommended for all the tested tissues and stages in B. germanica, whereas the combination of multiple reference genes was unfavorable in P. americana. This study provides a condition-specific resource of reference gene selection for accurate gene expression profiling and facilitating functional genomics in these two important cockroaches.
Highlights
The Blattaria cockroaches have evolved as an ancient and highly successful form of insect life. Some species in this group serve as public health pests, of which the German cockroach, Blattella germanica, and the American cockroach, Periplaneta americana, are the most common and troublesome household pests worldwide [1]
Each of the obtained primer pairs for quantitative real-time PCR (qPCR) amplification yielded a single peak from the melting fluorescence and a single band of the expected size in agarose gel (Figure S1), suggesting the absence of any non-specific product
A standard curve was established for each primer pair
Summary
The Blattaria cockroaches have evolved as an ancient and highly successful form of insect life. Some species (less than 1%) in this group serve as public health pests, of which the German cockroach, Blattella germanica, and the American cockroach, Periplaneta americana, are the most common and troublesome household pests worldwide [1] These two species are strictly synanthropic and usually infest human-built structures, including homes, apartments, restaurants, hospitals, and other places where food is available. They have long served as important models for studying chemical ecology, especially in the aspects of sex and aggregation pheromones [15,16,17,18,19,20] These omnivorous cockroach species are wildly used for examining host-gut microbiota interactions with regard to their development and behavior [21,22,23,24]
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