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.

Trypanosomatids (Euglenozoa: Kinetoplastea: Trypanosomatidae) are obligate parasitic flagellates with a worldwide distribution. They parasitize a wide range of vertebrates, invertebrates, and plants (Wallace 1966, Jaskowska et al. 2015, Correa et al. 2020, Votýpka et al. 2020, Frolov et al. 2021). The family Trypanosomatidae is divided into two nontaxonomic groups based on the number of hosts needed to complete development: monoxenous, requiring a single host (predominantly an insect), and dioxenous, requiring two distinct hosts, one usually an insect, the other, a vertebrate or plant (Correa et al. 2020, Malysheva et al. 2020, Frolov et al. 2021). The family Trypanosomatidae consists of 24 genera, of which 19 are monoxenous (Frolov et al. 2021). Worldwide, more than 10% of true bugs and flies are infected with monoxenous trypanosomatids and those found in the orders Diptera and Hemiptera: Heteroptera account for greater than 90% of the family diversity (Lukeš et al. 2018). Additional insect orders and suborders identified as hosts for trypanosomatids include Psocodea: Phthiraptera, Orthoptera, Hemiptera: Auchnorrhyncha, Hemiptera: Sternorrhyncha, Mecoptera, Hymenoptera, Siphonaptera, and Blattodea (reviewed in Frolov et al. 2021, Kostygov et al. 2021).

Within the hemipteran suborder Heteroptera, several species in the stink bug family Pentatomidae have been identified as hosts for trypanosomatids. These include the southern green stink bug Nezara viridula (L.) (Gibbs, 1957, Jankevicius et al. 1989, Fuxa et al. 2000), the neotropical brown stink bug Euschistus heros (F.) (Batistoti et al. 2001), the redbanded stink bug Piezodorus guildinii (W.) (Batistoti et al. 2001), and the brown marmorated stink bug Halyomorpha halys (Stål) (Malysheva et al. 2020).

Bagrada hilaris (Burmeister, 1835) is native to Africa, India, Asia, and the Middle East (Howard 1907, Husain 1924). First reported in 2008 in southern California (Arakelian 2008), it has spread rapidly throughout several southwestern U.S. states, Hawaii (Matsunaga et al. 2019) and Minnesota (Koch et al. 2018). It also has spread into Mexico ( Conti et al. 2021) and has been introduced into Chile (Faúndez et al. 2016, 2017; Carvajal et al. 2019). B. hilaris primarily feeds on plants in the family Brassicaceae (Cruciferae) but has a relatively large host range, feeding and damaging agriculturally important food crops including corn, kale, arugula, sunflower, among others (Reed et al. 2013).

During physiological investigations of B. hilaris, a flagellated protozoan was discovered in the alimentary canal. This manuscript characterizes the morphology and molecular identification of a trypanosomatid, which resembles those identified in other stink bug species. We tentatively identify this as a species in the genus Blastocrithidia. While little is known about monoxenous trypanosomatids in general, the possibility that not only an invasive stinkbug is now expanding its distribution in North America but that a new species of a potentially invasive trypanosomatid has also been introduced with unknown consequences makes this research valuable.

Materials and Methods

Insect Colony Origin and Maintenance

Bagrada hilaris of various ages were collected near the University of California campus (Riverside, CA), Gustine, CA, and Dixon, CA, from different plant species including London rocket, shortpod mustard, or sweet alyssum. Live specimens were shipped overnight to the USDA-ARS National Biological Control Laboratory (NBCL) in Stoneville, MS, for colony establishment (Grodowitz et al. 2019). Insects were housed in plastic or screen cages and fed varying combinations of broccoli florets or seedlings of other Brassicaceae. Plants were replaced with fresh material as needed.

Insect Dissections and Viewing

Individuals used to determine trypanosomatid presence, molecular studies, and morphological identifications were selected randomly and the dorsal abdominal cuticle removed to reveal the internal organs using methods previously published (Rojas et al. 2017, Grodowitz et al. 2019). Internal organs were extracted, and immediately or after a short duration (24–48 h) were placed in working buffer. This buffer consisted of 10% heat-inactivated Fetal Bovine Serum (Gemini Bio, Sacramento, CA) in TNM-FH Insect Medium (Gemini Bioproducts, Sacramento, CA) with 1 ml/20 ml gentamicin sulfate solution 100 mg/ml (Starhawk Laboratories, Shawnee Mission, KS) and 10 µl/20 ml Itraconazole (Sigma–Aldrich, Saint Louis, MO). Following the working buffer treatment, the organs were placed in phosphate-buffered saline (PBS) on a microscope slide. In some cases, gut contents were deposited on a poly-lysine coated microscopic slide (Thermo-Fisher Scientific, Miami, OK), fixed in 100% methanol for 2 min, and stained with Giemsa stain using manufacturer’s recommendations (Ricca Chemical, Arlington, TX).

Microscopes

To determine the presence of trypanosomatids in internal organs, primarily the alimentary canal, a variety of stereo and compound microscopes were used including the Leica M165C (removal of organs during dissection at 7–25×) or the Leica DMi1 (in association with the Neubauer hemocytometer (see section on number of trypanosomatids) (Leica Microsystems, Buffalo Grove, IL), Keyence DVX 5000 (Keyence Corporation, Itasca, IL), Nikon Eclipse E600 (Nikon Corporation, Tokyo, Japan), at magnifications ranging from 7× to 1,000× for elucidating morphological characteristics and presence of ‘straphanger’ cysts on the flagella. A high-speed video camera (Fastec TS5, Tech Imaging Services Inc., Saugus, MA) was used in conjunction with the Nikon Eclipse E600 to capture slow-motion video. Individual frames from the captured videos were used for morphological characterization and identifying potential reproductive modes.

Alimentary Number and Location of Trypanosomatids

Trypanosomatid density was determined using a Neubauer hemocytometer in combination with an inverted compound scope; the Leica DMi1 at 400x The alimentary canal was removed from ten randomly selected adults (five females and five males) at magnifications ranging from 7× to 25× using a Leica M165C, placed into PBS, homogenized using a small glass rod for 30 sec to dislodge trypanosomatids, and subsequently counted using a 10 µl aliquot of the homogenized gut. Reproductive trypanosomatids were defined as those possessing one or more cyst-like amastigotes or ’straphangers’ located on the flagella (Maslov et al. 2010). Non-reproductive trypanosomatids were devoid of cyst-like amastigotes. The presence of trypanosomatids were determined from the three main sections of the gut (fore, mid, and hind) from 29 randomly individuals though only 19 were found to contain trypanosomatids (eight males and eleven females).

Genome Sequencing, Assembly, and Analysis

Multiple wash buffer tubes were pooled and used to sequence 218,540,414 Illumina PE150 genomic reads (available at BioProject PRJNA661189), which were processed as described in Fig. 5 and in Supp Materials (online only). Using BLASTn (Altschul et al. 1990), assembly results were compared with the 69 trypanosomatid 18S rRNAs listed in Fig. 5 of Hughes and Piontkivska (2003), and matches were further compared with NCBI NT to aid species identification. Reads were aligned to Blastocrithidia papi (KX641338.1) and Blastocrithidia triatomae (KX138599.1) 18S rRNAs with Burrows-Wheeler Aligner (Li and Durbin 2009) and visualized in the Integrative Genomics Viewer (Robinson et al. 2011) to identify SNPs and/or indels. The process was repeated using assembled 18S rRNA fragments MT957144 and MT957145 (see below) as templates, to identify polymorphisms in the sequence data not reflected by the assembly consensus.

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.

Results and Discussion

During experimentation with B. hilaris to develop a physiological age-grading system (Grodowitz et al. 2019), large numbers of flagellated protozoans were observed within the alimentary canal. Based on morphology and previous reports of flagellated protozoans in the gut of other stink bugs (Fuxa et al. 2000, Batistoti et al. 2001), we identified these as trypanosomatid epimastigotes of a yet unidentified species. Numbers in the alimentary canal exceeded 100,000 per gut (106,700 ± 19,727 SE, n = 10) with close to 50% considered reproductive, i.e., containing one or more ‘straphanger’ cysts attached to the flagellum (44.5% ± 6.62 SE, n = 10). In some cases, we observed that trypanosomatids appeared to be attached perpendicularly to the midgut wall (Fig. 1a). Similar attachment was observed for Blastocrithidia papi, a trypanosomatid associated with the firebug, Pyrrhocoris apterus (L. 1758) (Frolov et al. 2017). However, this was not always the case for B. hilaris, and, in many instances, trypanosomatids appeared to be free-swimming within the gut. The epimastigotes were elongate with a single long, slender flagellum arising anteriorly (Fig. 1b), with the posterior portion often projected into a needle-like appendage, though this was not always present. No undulating membrane was observed. The mean distance from the centrally located nuclei to the kinetoplast was 9.08 µm ± 0.45 (mean ± SE, n = 19). The nuclei position in relation to the anteriorly situated kinetoplast, located just adjacent to the nuclei, indicates that this was an epimastigote even though no undulating membrane was observed (Wallace et al. 1983).

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.

Within the alimentary canal of many specimens, structures that appeared to be cyst-like amastigotes were found attached to the flagellum (Fig. 2a and b); these varied in size and numbers from one to five. In most cases, the cyst-like amastigotes appeared attached directly to the flagellum, though in a few cases, cyst-like amastigotes were attached by a slender thread-like structure (Fig. 2a). Maslov et al. (2010) termed cyst-like amastigotes attached in this manner ‘straphanger’ cysts. Current taxonomy of trypanosomes places cyst forming trypanosomatids into one of two groups; either the genus Blastocrithidia or a closely related clade with species related to Leptomonas jaculum (Kostygov and Frolov 2007, Votýpka et al. 2012, Záhonová et al 2016). This allows a further narrowing of the B. hilaris trypanosomatid identification.

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.

Trypanosomatids often were observed in a distinct formation where one larger epimastigote appeared attached to a central core of smaller flagellated individuals on the anterior portion of the flagellum forming what appeared to be a rosette (Fig. 2c and d). Frolov et al. (2017) described these as several smaller epimastigotes uniting with intertwined flagella. The formation of such a rosette structure in the alimentary canal of insects is common (Frolov et al. 2017); examples include several trypanosomatid species from different hemipteran species collected in Costa Rica (Frolov et al. 2017) and H. halys (Malysheva et al. 2020).

Photomicrographs of the entire alimentary canal of B. hilaris including the foregut, midgut, hindgut, and salivary glands are shown in Fig. 3. Of the 29 individuals examined to determine location of the trypanosomatids 19 individuals were found to contain trypanosomatids. Of these 19, trypanosomatids were found in the midgut 100% of the time with about half that number observed in either the foregut or hindgut (Fig. 4). While salivary glands were examined in only a few individuals, no trypanosomatids were detected. It has been reported that upon ingestion with food, the majority of trypanosomatids quickly transit through the insect foregut colonizing the mid- and hindgut (Malysheva et al. 2020, Frolov et al. 2021). Among monoxenous trypanosomatids, the single known exception is Herpetomonas nabiculae, of the predatory damsel bug Nabis flavomarginatus (Frolov et al. 2021).

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.

Morphology at the light microscopy level did not yield a definitive identification; thus, DNA sequencing was employed. The metagenomic assembly totaled 755,384,580 bases assembled into 397,539 scaffolds, and 476,723,366 bases in 826,020 contigs; the composite assembly N50 was 1,646 bp (Fig. 5). Two assembled sequences, available from NCBI as MT957144 and MT957145, are fragmentary and match with 100% identity to 18S rRNAs from Bl. papi (KX641338.1) and Bl. triatomae (KX138599.1; see Supp Fig. S1 [online only]). With respect to the Bl. papi exemplar, a 175 bp gap exists between MT957144 and MT957145, and with Bl. triatomae, 174 bp. Alignment of unassembled reads in these gaps demonstrates presence of two distinct haplotypes consistent with these Blastocrithidia species; their presence arises due to either intraspecific variation or the presence of multiple species. Read alignment to the MT957144 and MT957145 consensus sequences indicates additional, novel haplotypes in the data, rendering species identification incomplete.

The metagenomics assembly produced from this environmental sample, specifically the gut contents of B. hilaris, suggests the presence of one, or possibly more trypanosomatid species from the Blastocrithidia genus. Sequence data indicate these are, if not conspecific, at least highly similar to Bl. papi, a species isolated from Pyrrhochoris apterus (Linnaeus, 1758) (Hemiptera: Pyrrhocoridae) (Frolov et al. 2017), and Bl. triatomae, observed in Triatoma infestans (Klug) (Hemiptera: Reduviidae) (Schaub 1990). However, the presence of polymorphisms among read data creates a barrier to definitive species-level identification.

Illumina genome sequencing was pursued with the intent to analyze the protein coding content of trypanosomatids harbored in the B. hilaris alimentary canal as a beginning step in identifying to genus or even to species the trypanosomatid observed in B. hilaris. Other molecular studies are ongoing to confirm the identification and phylogeny.

The observation of trypanosomatids in the gut of B. hilaris is interesting though trypanosomatids in the Pentatomidae are not unusual being found in many different species (Votypka et al. 2012). Further research is warranted including trypanosomatid transmission, impact on host, life cycle, presence in other organs such as the Malpighian tubules, collection of higher quality genetic material to verify species ID and if multiple trypanosomatid species are present, culturing, among other. Understanding the role trypanosomatids play in the physiology of B. hilaris could lead to a better understanding of monoxenous trypanosomatids in pentatomids and potentially lead to advances in new strategies of control for B. hilaris and other invasive pentatomids.