Observations and collections were made in Marymoor Park, Redmond, WA (King County), at the head of the Sammamish River at the north end of Lake Sammamish. The site is located on the northern end of an off-leash dog area of the park and comprises a fairly large stand of P. emarginata trees bordered by a gravel parking lot and an open grassy field (47.6599, -122.1114). We visited the site periodically between 2015 and 2020. At each visit the area was surveyed for sawflies, and a selection was collected. We also looked for evidence of leaf mines at these visits. Adult specimens were identified in the lab using Smith (1971).

Oviposition of S. histrionica was assessed by collecting leaves at varying heights of P. emarginata. In 2015, leaves were collected by haphazardly selecting four trees in the stand and clipping branches from three heights: low (<0.75 m), medium (approximately 2 m), and high (2.5–3 m). In 2020 we repeated this sampling with two modifications. Trees were sampled on three transects through the stand: one along the north side, one through the center and one along the south side. Collections were made from every tree within 1 meter of each transect, by haphazardly selecting a single branch from each of four heights - < 1 m, 1.5 m, 3 m, and ≥ 5 m from the ground. In both years every collected leaf was examined, the number of eggs or mines was counted, and their location on the leaf was recorded. Egg or mine location was approximated by dividing the leaf into thirds, (basal, middle, apical), and indicating if the egg was on the margin, along a vein, or along the midvein.

We photo-documented mines at the collection site between 30 May and 15 June, 2018. On 15 May, 2020, we collected several S. histrionica adults and transferred them to Olympia WA, where they were placed in six groups of three (1 female to 2 males) in mesh bags on short branches (~15 cm) of potted P. emarginata plants. Two of the six groups resulted in successful oviposition of 5 and 9 eggs, respectively. The number of eggs on each leaf was counted and the location on the leaf was recorded. After the eggs hatched, mines were examined twice a week to track development. Larvae were sacrificed periodically and preserved in 70% EtOH. Upon each sacrificial event the mine was dissected and searched for head capsules to determine the number of instars up to that point. Measurements of larvae (n = 30) and discarded head capsules (n = 29) found within captive-reared (n = 9) and field-collected mines (n = 50) were made under a Leica MXC microscope using the Leica image software package. Five larvae were left to develop in the bagged mines.

Two specimens from the Marymoor collections, one larva and one adult, were selected for mitochondrial cytochrome oxidase subunit I (COI) sequencing sequencing. We were also provided with a single dead larva from a leaf mine on Prunus subcordata Benth. (Klamath plum) collected near Bass Lake, California, in June of 2021 (V. Albu 2021, pers. comm.). DNA from the whole larval bodies, and three legs from the adult, was extracted in 5 µl to 30 µl extraction volumes with 10% Chelex, and 4% Proteinase K (20 mg/mL). Samples were incubated at 56 °C for 1 hour, then at 99 °C for 20 min. Each 25 µl PCR reaction contained 5 µl of molecular grade water, 12.5 µl of 2X Platinum II Hot-start Green PCR Master Mix (Invitrogen), 1.25 µl of MgCl₂ (50 mM), 0.5 µl of F and R primers (10 µM), and 2 µl of DNA template. For the larval extraction, the primer pair LCO1490 and HCO2198 (Folmer et al. 1994) with M13 tails amplified the COI barcode region with cycling conditions as follows: initial denaturation at 95 °C for 1 min., 35 cycles of denaturation at 96 °C for 2 seconds, annealing at 50 °C for 5 seconds, and extension at 72 °C for 20 seconds. Final extension was at 72 °C for 2 minutes. Because adult sample PCR amplification using above primers was unsuccessful, the adult extraction was amplified using primer pair LepF1 and LepR1 (Hebert et al. 2004) with cycling conditions as follows: initial denaturation at 94 °C for 3 min., 40 cycles of denaturation at 94 °C for 20 seconds, annealing at 50 °C for 20 seconds, and extension at 72 °C for 30 seconds. Final extension was at 72 °C for 5 minutes. Amplification controls included a non-template control containing molecular grade water, and a positive control containing Lymantria dispar dispar DNA. Products were visualized via gel electrophoresis using a 1.5% agarose + 1X TBE gel at 160 Volts for 35 minutes. Amplicons of expected size were purified enzymatically using ExoSAP-IT Express (Applied Biosystems) kit per the manufacturer’s protocol. Samples were sequenced in both forward and reverse directions using the Big Dye Xterminator cycle sequencing kit v3.1 and BigDye Xterminator BDX clean up kit (Applied Biosystems) on the ABI SeqStudio following manufacturer guidelines. All sequences were manually trimmed for quality to 658 bp using Geneious Prime 2021.2, then aligned using MUSCLE 3.8.425 with 10 iterations. All resulting sequences were submitted to NCBI repository (Washington: ON181656 and ON181654, California: ON181655).

Setabara histrionica appears to be univoltine. Adults were active for a period of about 2 weeks during the spring, based on weekly visits to the site between mid-April and late May in 2018, 2019, and 2020 (Table 1). The earliest observations of adults were made on 5 May, 2014, and the latest were 23 May, 2018. Most adults were observed and captured in the understory, although a few adults were also collected by sweeping taller P. emarginata within reach (~3m). Over 2/3 of the specimens captured were males, and males outnumbered females at every collecting event except one.

The first mines became visible about two weeks after adult activity was observed. Because of the semi-cryptic nature of leaf-mining larvae, the precise number of days spent in each developmental stage was not recorded. The average number of days from egg hatch to final instar in our captive sawflies was 41 (n = 5). All of these specimens were lost during the final instar. It seems likely that the larvae escaped the mesh rearing bags, perhaps by chewing their way through or by squirming through small creases where the bag was secured to the Prunus limbs. Soil from the captive pots was sieved and examined, but no pupae or pre-pupae were located.

We collected 61 branches and 489 leaves in 2015, and recorded the location of 292 eggs. In 2020 we collected 114 branches and 1201 leaves, but located only 36 eggs. The majority of eggs discovered in both years were located in the lower tree branches (Table 2), with 61% were found within 1 m of the ground. All observed eggs were deposited on the margin of the leaf. Egg frequency decreased from the leaf stem towards the apex of each leaf, with more eggs located in the basal third than the central third, and more in the central third than in the apical third (Table 2).

The habitus is typical of leaf-mining Heterarthrinae (Smith 1971): head and body slightly dorsoventrally flattened, prolegs reduced, prognathous. Body white to cream-white. Head capsule slightly wider than long. Larval measurements are reported as averages.

Egg: less than 1 mm in diameter, round or slightly oblong, appearing as a light green blister on the outside of the leaf (Fig. 1).

Figure 1. 

Location of Setabara histrionica eggs on a Prunus emarginata leaf, and close up of egg.

First instar: body length (n = 3): 2.38 mm, head capsule width (n = 13): 0.38 mm. Head capsule beige to amber, thoracic legs white. Prolegs present on abdominal segments 2–8.

Second instar: body length (n = 2): 2.59 mm, head capsule width (n = 12): 0.51 mm.

Third instar: body length (n = 12): 5.06 mm, head capsule width (n = 18): 0.63 mm.

Final instar: body length (n = 17): 6.70 mm, head capsule width (n = 16): 0.75 mm (Figs 2, 3). Head dark brown, body mostly white with various dark plates. Head: Head capsule dark brown, lighter ventrally and around mouthparts; antenna 2-segmented; each mandible with 3 sharp apical teeth; maxillary palpus with 4 palpomeres, lacinia with row of about 10 subequal short spines; epipharynx with transverse row of about 10 spines on each half; labial palpus with three palpomeres. Thorax: Thoracic legs, large rectangular plate on pronotum, narrow plate on anterior margin of mesonotum, large rectangular plate on prosternum, small central plates on mesosternum and metasternum dark brown; thoracic legs 5 segmented; coxae without tubercles; tarsal claws present, simple. Abdomen: Small central dark brown plate on sternum of first segment (smaller than metasternal plate); prolegs present on segments 2–8 and 10, each proleg with anterior crescent-shaped dark spot, anal proleg with complete, circular dark spot; sometimes tiny supraspiracular spot present.

Figure 2. 

Fourth instar, head.

Figure 3. 

Fourth instar, ventral/dorsal view.

In Smith’s 1971 key to Heterarthrinae larvae, Setabara goes to couplet 6 which includes Messa (now Fenusella) and Profenusa (in part). The key can be amended as follows:

Mine: After hatching, the larva feeds on leaf tissue in a semi-circular shape moving away from the leaf margin (Fig. 4). Mines continue to be excavated in a blotch pattern, often constrained by the midvein of the leaf, unless near the thinner area at the apex. Frass was distinctly oblong and capsule-shaped, and was broadly dispersed throughout the mine (Fig. 5). The average area at larval maturity was 15.49 mm² (n = 11). Pre-pupae exited the mine through a chewed arced slit in the cuticle, which measured 2.13 mm on average (n = 4) (Fig. 6).

Figure 4. 

Mines at different stages of development.

Figure 5. 

Frass shape and distribution, taken from late instar mine.

Figure 6. 

Pre-pupal emergence hole.

The Washington specimens collected in this study were compared with two 421 bp COI sequences from S. histrionica specimens housed at the US National Museum of Natural History (USNM), also collected from Marymoor Park (BioProject PRJNA540960, GenBank accessions MW983660 and MW982369). All four trimmed Washington sequences were 100% identical to one another. In contrast, the sequence from the California larva was 95.3% identical to these, with 29 non-identical sites.