Deep Characterization of the Microbiomes of Calophya spp. (Hemiptera: Calophyidae) Gall-Inducing Psyllids Reveals the Absence of Plant Pathogenic Bacteria and Three Dominant Endosymbionts

Abstract

Bacteria associated with sap-feeding insect herbivores include not only symbionts that may increase their hosts’ fitness but also harmful plant pathogens. Calophya spp. gall-inducing psyllids (Hemiptera: Calophyidae) are being investigated for their potential as biological control agents of the noxious weed, Brazilian peppertree (Schinus terebinthifolia), in Florida. Although there are no examples of plant pathogen transmission by members of the family Calophyidae, several insects in the superfamily Psylloidea are known to transmit pathogenic bacteria in the genera Candidatus Liberibacter and Candidatus Phytoplasma. To determine whether Calophya spp. harbor potentially harmful plant pathogenic bacteria, we sequenced small subunit (SSU) ribosomal RNA (rRNA) gene amplicons generated from individuals from four Calophya spp. populations. All microbial SSU gene sequences fell into the bacterial domain, with 98-99% belonging to the Proteobacteria. The Calophya microbiomes contained a relatively simple community, with 49-79 operational taxonomic units (OTUs; 97%) detected, and only 5-8 OTUs with greater than 1% abundance. Candidatus Carsonella showed the highest relative abundance, with OTUs from this candidate genus representing between 51 – 65% of all recovered sequences. The next most abundant clade observed was an unclassified Enterobacteriacae group closely related to bacteria from the genera Buchnera and Blochmannia that ranged from 20-31% in relative abundance. Wolbachia populations were the third most abundant group and represented 7-27% of the diversity in microbial OTUs. No SSU rRNA gene sequences from putative pathogenic bacteria from the genera Ca. Liberibacter or Ca. Phytoplasma were detected in the microbiomes of the four Calophya populations. The probability that infected psyllids were present in our colonies, but were not sampled, was extremley low (1.39 x 10-10). As far as we are aware, our study is the first to characterize the microbiome of a candidate biological control agent, and coupled with previous work demonstrating a high degree of host specificity and absence of plant viruses, suggests that releasing Calophya spp. in United States poses minimal risk to non-target plants.

Inside out: microbiota dynamics during host-plant adaptation of whiteflies

“…B. tabaci is a phloem-feeding insect pest characterized by high fecundity, short developmental time, significant dispersal capability, and ability to use multiple plants as hosts [11]. Previous culture-independent approaches have reported that B. tabaci, as other sternorrhynchan insects (psyllids and aphids), presents low (if any) variety of gut-associated ectosymbionts [10,12,13]. This low diversity was mainly attributed to the sternorrhynchan simple gut morphology, the nearly microbial-free diet (phloem), the ability of the insects to digest the food quickly, and the presence of intracellular bacteria, also called endosymbionts, that might compete for the host resources [6,12,13].…”

“…Previous culture-independent approaches have reported that B. tabaci, as other sternorrhynchan insects (psyllids and aphids), presents low (if any) variety of gut-associated ectosymbionts [10,12,13]. This low diversity was mainly attributed to the sternorrhynchan simple gut morphology, the nearly microbial-free diet (phloem), the ability of the insects to digest the food quickly, and the presence of intracellular bacteria, also called endosymbionts, that might compete for the host resources [6,12,13]. All whiteflies, including B. tabaci, harbor an obligatory endosymbiont named Portiera aleyrodidarum and different facultative endosymbionts, including the genera Hamiltonella, Arsenophonus, Rickettsia, Wolbachia, and Cardinium [11,14].…”

The influence of bacteria on multitrophic interactions among plants, psyllids, and pathogen

“…C. ruddii," psyllids can associate with other bacteria as revealed by several microbial surveys. In some of these studies, bacterial communities associated with psyllids were not very diverse (Subandiyah et al, 2000;Nachappa et al, 2011;Raddadi et al, 2011;Fagen et al, 2012;Overholt et al, 2015), while other surveys found a greater diversity of bacteria associated with psyllids (Hail et al, 2012;Arp et al, 2014). Subsequent studies identified potential roles of secondary endosymbionts in psyllid reproduction, climate and host adaptation, and pathogen transmission.…”

“…For example, Wolbachia were found associated with several populations of D. citri in Florida (Subandiyah et al, 2000;Hert et al, 2008;Fagen et al, 2012;Saha et al, 2012;Hoffmann et al, 2014;Chu et al, 2016), China Ma et al, 2012), Iran and Pakistan (Lashkari et al, 2014), and Brazil (Guidolin & Consoli, 2013). Wolbachia were also found associated with populations of B. cockerelli (Nachappa et al, 2011;Hail et al, 2012;Arp et al, 2014), Cacopsylla pruni (Duron, 2013) and with nonvector gall-inducing psyllids in the genus Calophya (Hemiptera: Calophyidae) (Overholt et al, 2015).…”

Phylogenetic analyses and characteristics of the microbiomes from five mealybugs (Hemiptera: Pseudococcidae)

“…These methods are based on the deep sequencing of PCR-amplified bacterial 16S rRNA gene fragments, and the sequence data generated by HTS techniques are usually grouped into the same operational taxonomic unit (OTU) based on sequence similarity (≥97%), enabling the detection of all bacterial species existing in various samples. OTU is an effective tool for exploring bacterial communities, such as the symbiosis in aphids and psyllidae (Gauthier, Outreman, Mieuzet, & Simon, 2015;Hao & Chen, 2012;Jousselin et al, 2016;Overholt, Diaz, Rosskopf, Green, & Overholt, 2015). Therefore, HTS techniques are the first choice for studying the diversity of microbial communities (Degnan & Ochman, 2012).…”