Trypanosomatids Associated in the Alimentary Canal of Bagrada hilaris (Hemiptera: Pentatomidae)

Abstract

Bagrada hilaris (Burmeister) is an invasive pest of economically important crops in the United States. During physiological investigations of B. hilaris, a flagellated protozoan was discovered in the alimentary canal of many specimens. This manuscript characterizes the morphology and molecular identification of the trypanosomatid, which appears similar to trypanosomatids identified in other stink bug species. It has been identified as a species in the Blastocrithidia genus based on morphological characteristics and molecular analyses.

Keywords: Blastocrithidia; endosymbionts; stink bug.

Fig. 1.

(a) Photomicrograph frame captured from high-speed video footage showing large numbers of trypanosomatids from the midgut of Bagrada hilaris. Note that the trypanosomatids are mostly oriented perpendicular to the midgut wall; (b) photomicrograph of trypanosomatids found in the midgut of Bagrada hilaris and stained with Giemsa stain to differentiate the organelles. Note that the nucleus is adjacent to the kinetoplast and the kinetoplast is anterior to the nucleus. Only one flagellum is present and there is no undulating membrane apparent.

Fig. 2.

(a, b) Photomicrographs of trypanosomatids found in the gut of Bagrada hilaris containing ‘straphanger’ cysts attached to the flagellum—note (a) was captured from high-speed video footage, while (b) was from a slide of gut contents stained with Giemsa. (c, d) Photomicrographs of the formation of a rosette structure consisting of a single large epimastigote with a rosette shaped cluster of smaller individuals located at the anterior of the flagellum.

Fig. 3.

Photomicrograph of the alimentary canal of Bagrada hilaris including the foregut, midgut, hindgut, and salivary glands. The lines (black and white) running across the alimentary canal distinguish each of its major portions of the canal and are used to determine trypanosomatid presence in each distinct portion alimentary canal.

Fig. 4.

Percentage of times trypanosomatids were observed in each major section of the alimentary canal including the fore, mid, and hind gut.

Fig. 5.

Read cleaning, normalization, and assembly protocol. To augment basic quality-based read trimming initially performed by the sequencing vendor (Georgia Genomics and Bioinformatics Core, Atlanta, GA), quality trimming was done using Sickle (Joshi and Fass 2011). BBDuk of the BBTools suite (Bushnell 2016) was used for adapter trimming and contaminant filtering, and BBTools’ BBNorm was used for read normalization (target depth = 40, minimum depth = 2). Normalized reads were assembled into genomic contigs and scaffolds using the SOAPdenovo2 short-read assembler (k-mer parameter = 79, minimum contig length = 250 bp; Luo et al. 2012, 2015). Read pair counts at each step are shown; see Supp Table S1 (online only) for detailed information.