Diversity and localization of bacterial endosymbionts from whitefly species collected in Brazil

Abstract

Whiteflies (Hemiptera: Aleyrodidae) are sap-sucking insect pests, and some cause serious damage in agricultural crops by direct feeding and by transmitting plant viruses. Whiteflies maintain close associations with bacterial endosymbionts that can significantly influence their biology. All whitefly species harbor a primary endosymbiont, and a diverse array of secondary endosymbionts. In this study, we surveyed 34 whitefly populations collected from the states of Sao Paulo, Bahia, Minas Gerais and Parana in Brazil, for species identification and for infection with secondary endosymbionts. Sequencing the mitochondrial Cytochrome Oxidase I gene revealed the existence of five whitefly species: The sweetpotato whitefly Bemisia tabaci B biotype (recently termed Middle East-Asia Minor 1 or MEAM1), the greenhouse whitefly Trialeurodes vaporariorum, B. tabaci A biotype (recently termed New World 2 or NW2) collected only from Euphorbia, the Acacia whitefly Tetraleurodes acaciae and Bemisia tuberculata both were detected only on cassava. Sequencing rRNA genes showed that Hamiltonella and Rickettsia were highly prevalent in all MEAM1 populations, while Cardinium was close to fixation in only three populations. Surprisingly, some MEAM1 individuals and one NW2 population were infected with Fritschea. Arsenopnohus was the only endosymbiont detected in T. vaporariorum. In T. acaciae and B. tuberculata populations collected from cassava, Wolbachia was fixed in B. tuberculata and was highly prevalent in T. acaciae. Interestingly, while B. tuberculata was additionally infected with Arsenophonus, T. acaciae was infected with Cardinium and Fritschea. Fluorescence in situ hybridization analysis on representative individuals showed that Hamiltonella, Arsenopnohus and Fritschea were localized inside the bacteriome, Cardinium and Wolbachia exhibited dual localization patterns inside and outside the bacteriome, and Rickettsia showed strict localization outside the bacteriome. This study is the first survey of whitely populations collected in Brazil, and provides further insights into the complexity of infection with secondary endosymionts in whiteflies.

Figure 1. Individual and mixed infection by secondary symbionts in B.tabaci populations collected in this study.

Twenty two MEAM1 (B biotype) and one NW2 (A biotype) populations were collected and surveyed. Each box represents one population. Vertical columns represent the different symbionts tested as indicated in the base of each column, and each horizontal column represents one individual that was tested for the presence of the six different symbionts. Shaded boxes represent positive infection with the tested symbiont. The geographical origin of the populations, the species and the number of individuals tested are indicated at the top of each box (see Table 1 for full names).

Figure 2. Individual and mixed infections with secondary endosymbionts in additional whitefly species analyzed in this study.

Four populations of T. vaporariorum (T. v.), 4 populations of B. tuberculata (B. tub.) and 3 populations of T. acaciae (T. a.) were collected and analyzed in this study (See legend to figure 2 for more details).

Figure 3. FISH of selected specimens collected in this study for specific localization of secondary endosymbionts.

A–B: FISH of Arsenophonus (yellow) and Portiera (red) in T. vaporariorum under bright field (A) and dark field (B). C–D: FISH of Fritschea (blue) and Portiera (red) in NW2 under bright field (C) and dark field (D). E–F: FISH of Hamiltonella (green) and Portiera (red) in MEAM1 under bright field (E) and dark field (F). G–H: FISH of Rickettsia (blue) and Portiera (red) in MEAM1 under bright field (G) and dark field (H). I–J: FISH of Cardinium (blue) and Portiera (red) in T. acaciae under bright field (I) and dark field (J). K–L: FISH of Wolbachia (blue) in B. tuberculata under bright field (K) and dark field (L). T: testicle.