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Supplementary Tables 1-3. Supplemental Table 1. Plasma pharmacokinetics of DMS612 metabolites. Supplemental Table 2. Pharmacodynamic assessment of !-H2AX detection in PBMCs. Mean values for !-H2AX foci per cell with DMS612 doses of 1.5, 3, 5, 7, 9 and 12 mg/m2 at the indicated time in hours (h). Supplemental Table 3. Pharmacodynamic assessment of !-H2AX detection in hair follicles. Mean values for cell with more than 4 !-H2AX foci per cell in plucked scalp hairs with DMS612 doses of 1.5, 3 and 9 mg/m2 at the indicated time in hours (h).

All supplementary files together

Supplementary Tables 1-3. Supplemental Table 1. Plasma pharmacokinetics of DMS612 metabolites. Supplemental Table 2. Pharmacodynamic assessment of !-H2AX detection in PBMCs. Mean values for !-H2AX foci per cell with DMS612 doses of 1.5, 3, 5, 7, 9 and 12 mg/m2 at the indicated time in hours (h). Supplemental Table 3. Pharmacodynamic assessment of !-H2AX detection in hair follicles. Mean values for cell with more than 4 !-H2AX foci per cell in plucked scalp hairs with DMS612 doses of 1.5, 3 and 9 mg/m2 at the indicated time in hours (h). Supplemental Figure 1. Pharmacokinetics of DMS612 Metabolites.

Purpose: DMS612 is a dimethane sulfonate analog with bifunctional alkylating activity and preferential cytotoxicity to human renal cell carcinoma (RCC) in the NCI-60 cell panel. This first-in-human phase I study aimed to determine dose-limiting toxicity (DLT), maximum tolerated dose (MTD), pharmacokinetics (PK), and pharmacodynamics (PD) of DMS612 administered by 10-minute intravenous infusion on days 1, 8, and 15 of an every-28-day schedule.Experimental Design: Patients with advanced solid malignancies were eligible. Enrollment followed a 3+3 design. PKs of DMS612 and metabolites were assessed by mass spectroscopy and PD by γ-H2AX immunofluorescence.Results: A total of 31 patients, including those with colorectal (11), RCC (4), cervical (2), and urothelial (1) cancers, were enrolled. Six dose levels were studied, from 1.5 mg/m2 to 12 mg/m2. DLTs of grade 4 neutropenia and prolonged grade 3 thrombocytopenia were observed at 12 mg/m2. The MTD was determined to be 9 mg/m2 with a single DLT of grade 4 thrombocytopenia in 1 of 12 patients. Two patients had a confirmed partial response at the 9 mg/m2 dose level, in renal (1) and cervical (1) cancer. DMS612 was rapidly converted into active metabolites. γ-H2AX immunofluorescence revealed dose-dependent DNA damage in both peripheral blood lymphocytes and scalp hairs.Conclusions: The MTD of DMS12 on days 1, 8, and 15 every 28 days was 9 mg/m2. DMS612 appears to be an alkylating agent with unique tissue specificities. Dose-dependent PD signals and two partial responses at the MTD support further evaluation of DMS612 in phase II trials. Clin Cancer Res; 21(4); 721–9. ©2014 AACR.

Purpose:Only one chemical class of topoisomerase I (TOP1) inhibitors is FDA approved, the camptothecins with irinotecan and topotecan widely used. Because of their limitations (chemical instability, drug efflux-mediated resistance, and diarrhea), novel TOP1 inhibitors are warranted. Indenoisoquinoline non-camptothecin topoisomerase I (TOP1) inhibitors overcome chemical instability and drug resistance that limit camptothecin use. Three indenoisoquinolines, LMP400 (indotecan), LMP776 (indimitecan), and LMP744, were examined in a phase I study for lymphoma-bearing dogs to evaluate differential efficacy, pharmacodynamics, toxicology, and pharmacokinetics.Experimental Design:Eighty-four client-owned dogs with lymphomas were enrolled in dose-escalation cohorts for each indenoisoquinoline, with an expansion phase for LMP744. Efficacy, tolerability, pharmacokinetics, and target engagement were determined.Results:The MTDs were 17.5 mg/m2 for LMP 776 and 100 mg/m2 for LMP744; bone marrow toxicity was dose-limiting; up to 65 mg/m2 LMP400 was well-tolerated and MTD was not reached. None of the drugs induced notable diarrhea. Sustained tumor accumulation was observed for LMP744; γH2AX induction was demonstrated in tumors 2 and 6 hours after treatment; a decrease in TOP1 protein was observed in most lymphoma samples across all compounds and dose levels, which is consistent with the fact that tumor response was also observed at low doses LMP744. Objective responses were documented for all indenoisoquinolines; efficacy (13/19 dogs) was greatest for LMP744.Conclusions:These results demonstrate proof-of-mechanism for indenoisoquinoline TOP1 inhibitors supporting their further clinical development. They also highlight the value of the NCI Comparative Oncology Program (https://ccr.cancer.gov/Comparative-Oncology-Program) for evaluating novel therapies in immunocompetent pets with cancers.

A key to the known fourth instar larval stages of North American Metriocnemus van der Wulp 1. Mentum with 4 small median teeth and 5 pairs of lateral teeth (Epler 2001: page 7.86, couplet 2(1’))................................................................................................... M. knabi Coquillett, 1904 - Mentum with bifid or simple median tooth and 4 to 5 pairs of lateral teeth........................................ 2 2. Mentum with simple median tooth and 5 pairs of lateral teeth (Saether 1989: Figure 12G)...... M. ursinus (Holmgren, 1869) - Mentum with bifid median tooth and 4 to 5 pairs of lateral teeth................................................ 3 3. Mentum with a wide bifid median tooth and 4 pairs of lateral teeth (Fig. 3h)...................... M. erthranthei sp. nov. - Mentum with narrow bifid median tooth and 5 pairs of lateral teeth.............................................. 4 4. Median tooth of the mentum is comparatively small, sitting much lower than lateral teeth............................ 5 - Median tooth of the mentum comparatively well-developed, sitting slightly lower or higher than lateral teeth............ 6 5. Basal antennal segment no longer than wide (Saether 1989: Figure 12B). Labral lamella and SI simple (Saether 1989: Fig. 12D). Median tooth of mentum sunken (Epler 2001: page 7.86, couplet 3(2’); Orendt & Bendt 2021: page 119, couplet D7; Saether 1989: Fig. 12H). Anal tubules long and slender (Saether 1989: Fig. 12J)..................... M. fuscipes (Meigen, 1818) - Basal antennal segment longer than wide (Fig. 7b). Labral lamella comb-like and SI trifid (Fig. 5c). Median tooth of mentum not sunken (Fig. 7e). Anal tubules short and wide (Fig. 7f)......................................... M. sp. “ Oregon ” 6. Antenna 4 segmented (Cranston & Judd 1987: Fig. 4a). Basal antennal segment L/W> 4. Median tooth of mentum stands higher than 1 st lateral teeth. Setae submenti aligned with the 3 rd lateral teeth of mentum (Cranston & Judd 1987: Fig. 4c)............................................................................ M. yaquina Cranston & Judd, 1987 - Not with the above combination of characters............................................................... 7 7. Abdominal segments with long setae, each half as long as the segment bearing it.................... M. sp. A Epler, 2001 - Abdominal segments without long setae................................................................... 8 8. Procercus as wide as long or wider than long.AR 1.7–1.8. Basal antennal segment L/W: 2.3 (Th. Bendt & H.K.M. Moller Pillot, in litt.)................................................................................ M. tristellus agg. * - Not with the above combination of characters............................................................... 9 9. Median bifid tooth of mentum as high as the 1 st lateral teeth (Saether, 1989: Fig. 6E). Basal antennal segment L/ W 2.5 –2.6. Head length 0.44–0.45 mm................................................................. M. brusti Saether, 1989 - Median bifid tooth of mentum clearly lower than 1 st lateral teeth. Basal antennal segment L/ W 1.1 –2.4. Head length 0.13–0.47 mm............................................................................................... 10 10. Basal antennal segment L/ W 1.1. AR 0.67. Mandible with 3 inner teeth (Epler 2001: page 7.162). Procercus with 4 anal setae.............................................................. M. sp. (= Orthocladiinae genus E, Epler, 2001) ** - Not with the above combination of characters.............................................................. 11 11. AR 1.5–1.8. Head length 0.28-0.31 mm (Saether, 1989)............................... M. albolineatus (Meigen, 1818) - AR 2.0–2.2. Head length 0.39–0.47 (Saether, 1989)................................ M. eurynotus (Holmgren, 1883) ***, Published as part of Eiseman, Charles S., Namayandeh, Armin, Linden, John Van Der & Palmer, Michael W., 2023, Metriocnemus erythranthei sp. nov. and Limnophyes viribus sp. nov. (Diptera: Chironomidae: Orthocladiinae): leafminers of monkeyflowers, speedwells, and other herbaceous plants, with new observations on the ecology and habitats of other leaf-mining Chironomidae, pp. 41-68 in Zootaxa 5249 (1) on pages 56-57, DOI: 10.11646/zootaxa.5249.1.3, http://zenodo.org/record/7685232, {"references":["Epler, J. H. (2001) Identification manual for the larval Chironomidae (Diptera) of North and South Carolina. A guide to the taxonomy of the midges of the southeastern United States including Florida. North Carolina Department of Environment and Natural Resources Division of Water Quality, St. Johns River Water Management District, Palatka, Florida, iv + 530 pp.","Saether, O. A. (1989) Metriocnemus van der Wulp: a new species and a revision of species described by Meigen, Zetterstedt, Staeger, Holmgren, Lundstr ˆ m and Strenzke (Diptera: Chironomidae). Insect Systematics & Evolution, 19, 393 - 430. https: // doi. org / 10.1163 / 187631289 X 00528","Orendt, C. & Bendt, Th. (2021) Orthocladiinae sensu lato (Orthocladiinae, Prodiamesinae, Diamesinae, Podonominae, Buchonomyiinae, Telmatogetoninae) (Diptera: Chironomidae). Keys to Central European larvae with respect to macroscopic characters. DGL-Arbeitshilfe (DGL Tools) 1 - 2021. German Limnological Society (DGL) e. V., Essen, 140 pp.","Cranston, P. S. & Judd, D. D. (1987) Metriocnemus (Diptera: Chironomidae): an ecological survey and description of a new species. Journal of the New York Entomological Society, 95, 534 - 546."]}

Metriocnemus eurynotus (Holmgren, 1883) (Figs. 6–7) Material examined. USA: IOWA: Winneshiek Co., Decorah, Van Peenan Spring at Van Peenan Park, 43.312834, -91.776010, 14.iv.2022, em. 6.v.2022, leg. J. van der Linden, ex thallose liverwort (1Ô, 1 pupa, 1 larva, ANC); same location, 10.v.2022, preserved 16.v.2022, leg. J. van der Linden, ex Impatiens sp. (1 larva, USNM); same but preserved 21.v.2022 (2 pupae, ANC); same but em. 21.v.2022, (1♀, ANC); same but em. 23.v.2022 (2♀, 2 pupae, 2 larvae, ANC); same but em. by 27.v.2022 (2♀♀, ANC); same location, 17.v.2022, em. 21.v.2022, leg. J. van der Linden, ex Impatiens sp. (1Ô, 1 pupa, 1 larva, USNM); same but em. 23.v.2022 (1♀, ANC); same but em. by 27.v.2022 (2♀♀, 1 pupa, ANC); same location, 17.v.2022, leg. J. van der Linden, ex Veronica sp. (1 larva, USNM); same but em. 29.v.2022 (1♀, 2 pupae, 1 larva, USNM); OREGON: Lane Co., Blue River, 44.1535, -122.328, 3.vi.2022, leg. M. W. Palmer, ex Petasites frigidus (2 larvae, USNM). Biological notes. It appears that nothing has been published previously about the larval habits of this Holarctic species (Saether 1989, 1995). John van der Linden (JvdL) was examining 5- to 10-cm tall sprouts of jewelweed (Balsaminaceae: Impatiens sp.; very likely I. capensis Meerb.) on the margin of a rocky spring-fed pool in Iowa in early May when he noticed mines in cotyledons of some of the plants (Fig. 6a). Chironomid larvae were present inside the mines and on the surfaces of the cotyledons and appeared to move freely between these niches (Figs. 6b–h). The mines, which were only observed in Impatiens cotyledons and not in the developing true leaves, consisted of short, irregular tunnels into the tissue emanating from a central blotch. Tears or holes in one or both epidermises in the central blotch area allowed larvae to enter and exit the mines. In some older mines, the central area’s epidermis had fallen away completely, resulting in a hole in the cotyledon. In captivity, larvae were observed to move around in the mines and feed on mesophyll. The mines contained sparsely scattered green or greenish-brown pellets or rods of frass. One feeding larva with similarly colored material in its gut was observed depositing excrement in the mine. As many as five larvae were observed inside or on the edges of one mined area on a single cotyledon (video at https:// youtu.be/Tt4 SYMH 3U5U). Larvae interacted vigorously, especially upon physical contact or very close proximity, to which they responded with thrashing movements or by appearing to bite or nip each other’s bodies (Fig. 6i). Most captive larvae soon exited the mines permanently. They switched to feeding externally on the cotyledons (Fig. 6j). In containers holding multiple larvae, two or three individuals fed communally on a cotyledon and, in some cases, consumed it entirely before reaching maturity. Full-grown larvae pupated exposed on the cotyledon remnants or on the moist paper towel bedding or sides of the containers (Fig. 6k). Nine adults emerged (Fig. 6l). Several other individuals reached pupation. However, they failed to emerge as adults, with some darkened, mature pupae crawling around the rearing container actively, only to perish a short time later. Larvae of M. eurynotus were not observed to initiate new mines in pristine cotyledons. Although no other insects were observed feeding on or in them, we cannot exclude the possibility that there was some initial damage to the cotyledons that allowed the larvae to enter them. A week after the larvae were first discovered, more M. eurynotus larvae were found on leaves of Veronica sp. in the same rocky spring, both moving around on the leaf surfaces and feeding within leaf mines of M. erythranthei (Fig. 12a; videos at https://youtu.be/n9J6RJ0-DnI and https://youtu.be/qQ6k2eWBz_I). One male was also reared from a thallose liverwort collected from this site in midApril, although the larva was never actually observed in this case. There were mines in the liverwort, at least some of which appeared to be agromyzid in origin. It is conceivable that the M. eurynotus individual fed as a larva inside these. Before starting the rearing, the undersides and rhizoids of the thalli were thoroughly washed and massaged in water to remove the substrate. The pupa of this individual was found loose in the rearing container on 5 May, and examination of the thalli at this time revealed that a few of them showed evidence of heavy external feeding; no possible source for this was found other than the M. eurynotus, and the damage was similar in appearance to the external feeding on the jewelweed cotyledons. In Oregon, Mike W. Palmer (MWP) collected larvae of M. eurynotus on leaves of Petasites frigidus along with M. erythranthei and the Metriocnemus species discussed below., Published as part of Eiseman, Charles S., Namayandeh, Armin, Linden, John Van Der & Palmer, Michael W., 2023, Metriocnemus erythranthei sp. nov. and Limnophyes viribus sp. nov. (Diptera: Chironomidae: Orthocladiinae): leafminers of monkeyflowers, speedwells, and other herbaceous plants, with new observations on the ecology and habitats of other leaf-mining Chironomidae, pp. 41-68 in Zootaxa 5249 (1) on pages 51-54, DOI: 10.11646/zootaxa.5249.1.3, http://zenodo.org/record/7685232, {"references":["Saether, O. A. (1989) Metriocnemus van der Wulp: a new species and a revision of species described by Meigen, Zetterstedt, Staeger, Holmgren, Lundstr ˆ m and Strenzke (Diptera: Chironomidae). Insect Systematics & Evolution, 19, 393 - 430. https: // doi. org / 10.1163 / 187631289 X 00528"]}

Limnophyes viribus Namayandeh, Eiseman, Palmer & van der Linden sp. nov. (Figs. 9–11) LSID: urn:lsid:zoobank.org:act:53ef97e1-5c99-4e60-9847-d3e1e61aae0c Holotype. USA: OREGON: Lane Co., Blue River, 44.1535, -122.328, 5.iv.2022, em. 10–14.iv.2022, leg. M. W. Palmer, ex Erythranthe guttata complex (1Ô, USNM). Paratypes. USA: IOWA: Winneshiek Co., Decorah, Van Peenan Spring, Van Peenan Park, 43.312834, - 91.776010, 17.v.2022, em. 21.v.2022, leg. J. van der Linden, ex. Impatiens sp. (1Ô, USNM); OREGON: same data as holotype (5ÔÔ, 4♀♀, 1 larva, USNM). Other material examined. USA: OREGON: same data as holotype (9ÔÔ, 2♀♀, ANC). Etymology. The new species is named after a phoenix sculpture named “ Viribus,” which represents resiliency and rebirth, made by sculptor Jud Turner. The monument stands in the McKenzie River Corridor town of Blue River, the type locality, which was ravaged by the Holiday Farm Fire of 2020. The word is Latin and means “strength.” Description. Male (n = 9). Total length 1.8–2.0, 1.9 mm. Wing 1.3–1.4 mm long and 0.3–0.4 mm wide. Coloration. Head, thorax, legs, tergites, sternites, and hypopygium black. Wings and halters grey. Head. Antenna with 13 flagellomeres, last flagellomere with 6 sensilla chaetica, groove starts at third segment, AR 0.3–0.6, 0.5. Eyes bare, without dorsomedial extension. Temporal setae few, 2 outer verticals and 1 frontal. Tentorium 132–135, 133 μm long (Fig. 9a). Clypeus rectangular, 60 μm long and 114 μm wide, bearing 15 setae, setae 57–70, 63 μm long. Palpal segment lengths (in μm): 27–29, 28; 32–39, 36; 65–68, 66; 49–57, 53; 70–108, 89. Third palpomere with 1 sensilla clavata. Thorax (Fig. 9b). Acrostichals 4; dorsocentrals 20–25, around 23 in a single row and remainder in post humeral region double rows; prealars 6–7; scutellars 6–7 in single row; 3–6 lanceolate humerals; 6–8 lanceolate prescutellars; 10–14, 12 antepronotals; 5 posterior anepisternals II; 3 median anepisternals II; 2 epimeron II; 8 preepisternals, 6 anteriorly clustered and diagonal, separated from 2 vertical. Wing (Fig. 9c). Brachiolum with 1 seta. Squama bare. R with 11 setae, R 1 with 4 setae, other veins bare. Costa extension 45 μm. Anal lobe not projecting. Microtrichia visible at 10 ×. Legs. Fore tibia spur 37–45, 41 μm long, mid tibia spurs 20–24, 22 and 19 μm long, hind tibia spurs 42–50, 46 and 14–18, 16 μm long, hind tibia comb with around 12 spines. Lengths and proportions of legs as in Table 3. Hypopygium (Fig. 9d). Tergite IX with around 4 setae close to the base of anal point. Anal point extremely short, almost receded, wide with apex rounded; anal point 7–12, 10 μm long and 17–26, 21 μm wide. Virga consists of single long mid-spine with around 4 shorter lateral spines, the main spine 25–32, 28 μm long 7. Sternapodeme transverse with well-developed oral projections; sternapodeme 74–91, 81 μm long. Phallapodeme 33–42, 38 μm long. Inferior volsella a large triangular lobe with narrow apex; covered in numerous simple setae. Gonostylus large, expanded apically and with small spine-like distal outer projection, 60–77, 70 μm long; crista dorsalis large, overarching the apex of gonostylus. Gonocoxite 110–123, 116 μm long. HR 1.5–1.8, 1.7, HV 2.5–2.6. Female (n = 2). Total length 1.7–1.9, 1.8 mm. Wing 1.2 mm long and 0.37–0.43, 0.40 mm wide. Coloration. Same as the male. Head (Fig. 10a). Antenna with 5 flagellomeres, last flagellomere with 8 sensilla chaetica, 1 st –4 th segments each with 2 sensilla chaetica, AR 0.4–0.7, 0.5. Eyes bare. Temporal setae 3–4 including 2–3 outer vertical and 1 frontal. Tentorium 120–123, 122 μm long. Clypeus rectangular, 65–82, 74 μm long and 96–110, 104 μm wide, bearing 20 setae, setae 53–63, 59 μm long. Palpal segment lengths (in μm): 27–31, 29; 31–34, 32; 57–71, 64; 61; 72–90, 81. Thorax (Fig. 10b). Acrostichals 4; dorsocentrals 26, around 23 in a single row and remainder in post humeral region double rows; prealars 5; scutellars 7 in single row; 8 lanceolate humerals; 8 lanceolate prescutellars; 6 antepronotals; 4 posterior anepisternals II; 3 epimeron II; 8 preepisternals, 6 anteriorly clustered and diagonal, separated from 2 vertical. Wing (Fig. 10c). Brachiolum with 1 seta. Squama bare. R with 9–13, 11 setae; R 1 with 5–7 setae; R 4+5 12–15 setae; other veins without setae. Costa extension 27–33 μm. Microtrichia visible at 10 ×. Legs. Hind and mid femur with keel. Fore tibia spur 24 μm long, mid tibia spurs 18–20, 19 and 13–17, 15 μm long, hind tibia spurs 40–47, 45 and 16–17 μm long; hind tibia comb with around 12 spines. Lengths and proportions of legs as in Table 4. Genitalia (Figs. 10d–e). Seminal capsules comparatively large, 66–70, 68 μm long, and 49–57, 53 μm wide, semi-circular, spermathecal ducts with loops, with well-developed bulb (Fig. 10d). Notum 154–174, 158 µm long. Gonapophysis VIII divided into ventrolateral and thin dorsomesal lobe (Fig. 10d).Apodeme lobe distinct. Gonocoxite developed with around 8–9 setae (Fig. 10e). Tergite IX undivided. Cercus small, crescent-shaped, 60–61 µm long, and 31–41, 36 µm wide (Fig. 10e). Pupa. At present not known. Pupal exuviae could not be retrieved from the leaves. Larva (n = 1). Total length 3.0 mm. Head 231 μm long and 267 μm wide. Coloration (Fig. 11a). Head capsule yellow with postmentum region darker. Occipital margin much darker in contrast to the remainder of the head. Abdomen bluish grey with patches of white. Head. Antenna 5 segmented; segments length in μm: 30, 11, 2, 5, 3; ring organ closer to the apex of the basal segment; blade as long as the flagellum, blade 21 μm long (Fig. 11b); AR 1.4. Labral SI serrate, SII–SIII simple (Fig. 11c). Premandible wide and bifid, dark apically, 49 μm long (Fig. 11c). Mandible dark, apical tooth shorter than combined width of 3 inner teeth; seta subdentalis very small; setae interna with several very long branches (Fig. 11d), mandible 86 μm long. Mentum dark, with large bifid median tooth and 5 pairs of lateral teeth, median tooth 2.8 × the 1 st lateral teeth; seta submenti posteriad to mentum aligned with 2 nd lateral tooth (Fig. 11e); mentum 57 μm long and 68 μm wide; ventromental plate 39 μm long, and 14 μm wide, large, slightly reaching beyond the margin of mentum. Abdomen. Posterior parapods 65 μm long and 40 μm long, bearing around 12 simple dark claws (Fig. 11f). Procercus 23 μm long and 17 μm wide, bearing 6 apical setae, apical setae 217 μm long; supraanal setae 144 μm long; four anal tubules with constriction, anal tubules 91 μm long (Fig. 11f). Diagnostic characters. Limnophyes viribus can be separated from other related species by the combination of the following characteristics: The adult male is characterized by AR of 0.3–0.6; 3–6 lanceolate humerals; 6– 8 lanceolate prescutellars; 8 preepisternals, 6 anteriorly clustered and diagonal, separated from 2 vertical; anal point extremely short, almost receded; virga consists of single long mid-spine with around 4 shorter lateral spines; inferior volsella a large triangular lobe with narrow apex; gonostylus large, expanded apically with small spine-like distal outer projection. The adult female is characterized by AR of 0.4–0.7; 8 lanceolate humerals; 8 lanceolate prescutellars; 8 preepisternals, 6 anteriorly clustered and diagonal, separated from 2 vertical; seminal capsules comparatively large. The larva is characterized by AR 1.4; premandible wide and bifid; setae interna of the mandible with several very long branches; mentum with large bifid median tooth and 5 pairs of lateral teeth; procercus bearing 6 apical setae; supraanal setae long; anal tubules longer than posterior parapods. Taxonomic remarks. The adults of L. viribus resemble Limnophyes pilicistulus Saether, 1975. The two species are probably related and form a sister group. Adults of L. viribus can be separated from L. pilicistulus by the lower number of temporal setae, higher number of lanceolate humerals and prescutellars. Additionally, the adult male of L. viribus has a virga with more branches, and a gonostylus with more overarching crista dorsalis, and a small spinelike distal outer projection. The adult female of L. viribus has longer cercus and notum and shorter seminal capsules compared to L. pilicistulus. Biological notes. The larvae are apparently secondary inhabitants in leaf mines of Metriocnemus species, as with the undetermined Limnophyes species discussed below. The single Iowa specimen emerged in a batch rearing of M. eurynotus larvae feeding on Impatiens cotyledons. Four days earlier, in another rearing container, a mine with a larva of M. eurynotus had been found to also contain a much smaller larva that appeared uniformly dark and may have been an individual of L. viribus (photos at https://bugguide.net/node/view/2126834). Also in Iowa, a female very likely representing L. viribus was reared from a collection of M. erythranthei larvae mining Veronica leaves (see below under “ Limnophyes spp. ”). The Oregon specimens were reared along with M. erythranthei from plants of the Erythranthe guttata complex, although it was not entirely clear whether they emerged from leaves or the muck surrounding the roots., Published as part of Eiseman, Charles S., Namayandeh, Armin, Linden, John Van Der & Palmer, Michael W., 2023, Metriocnemus erythranthei sp. nov. and Limnophyes viribus sp. nov. (Diptera: Chironomidae: Orthocladiinae): leafminers of monkeyflowers, speedwells, and other herbaceous plants, with new observations on the ecology and habitats of other leaf-mining Chironomidae, pp. 41-68 in Zootaxa 5249 (1) on pages 57-61, DOI: 10.11646/zootaxa.5249.1.3, http://zenodo.org/record/7685232

Psectrocladius (s.s.) vernalis (Malloch, 1915) Material examined. USA: IOWA: Winneshiek Co., Decorah, Van Peenan Spring at Van Peenan Park, 43.312834, -91.776010, 17.v.2022, em. 22.v.2022, J. van der Linden, ex Veronica sp. (1Ô, USNM). Biological notes. The rearing container in which this specimen emerged included a single mined Veronica leaf inhabited by multiple Metriocnemus erythranthei larvae and one or two M. eurynotus larvae. The photographed M. eurynotus larva (Fig. 12a) exited the mine a short time later and began wandering on the leaf surface; it was later preserved. A second unmined Veronica leaf containing no larvae was added to the container at one point, and the M. erythranthei larvae began mining it after exiting their original leaf. The Psectrocladius vernalis male emerged five days after the original mined leaf was collected, along with a male of M. erythranthei. It would seem that it was one of the larvae feeding together in the mine and was not recognized as distinct from the M. erythranthei larvae. Larvae of P. vernalis have been found to live on the surfaces of submerged plants, including Eurasian milfoil (Haloragaceae: Myriophyllum spicatum L.) and native water stargrass (Pontederiaceae: Heteranthera dubia (Jacq.) MacMill.), but their habits were not observed (Balci & Kennedy 2003)., Published as part of Eiseman, Charles S., Namayandeh, Armin, Linden, John Van Der & Palmer, Michael W., 2023, Metriocnemus erythranthei sp. nov. and Limnophyes viribus sp. nov. (Diptera: Chironomidae: Orthocladiinae): leafminers of monkeyflowers, speedwells, and other herbaceous plants, with new observations on the ecology and habitats of other leaf-mining Chironomidae, pp. 41-68 in Zootaxa 5249 (1) on page 62, DOI: 10.11646/zootaxa.5249.1.3, http://zenodo.org/record/7685232, {"references":["Balci, P. & Kennedy, J. H. (2003) Comparison of chironomids and other macroinvertebrates associated with Myriophyllum spicatum and Heteranthera dubia. Journal of Freshwater Ecology, 18, 235 - 247. https: // doi. org / 10.1080 / 02705060.2003.9664490"]}

Metriocnemus sp. “ Oregon ” (Figs. 7a–f) Material examined. USA: OREGON: Lane Co., Blue River, 44.1535, -122.328, 3.vi.2022, leg. M. W. Palmer, ex Claytonia sibirica (4 larvae, USNM; 5 larvae, ANC); same but ex Myosotis scorpioides (1 larva, USNM); same but ex Petasites frigidus (2 larvae, USNM; 11 larvae, ANC). Larva (n = 7). Total length 4.5–6.0, 5.3 mm. Head 288–364, 339 μm long, 429–579, 458 μm wide. Coloration (Fig. 5a). Head capsule dark brown. Occipital margin much darker in contrast to the remainder of the head. Abdomen greenish yellow with a bluish pattern on the 1 st and 2 nd segments. Head. Antenna short, 5 segmented; antennal segments in μm: 43, 11–14, 3–4, 4–5, 5; 1 st antennal segment L/ W 1.2; ring organ located at mid-section of 1 st segment; AR 1.7–1.8, blade subequal to flagellum; blade 23–25, 24 μm long (Fig. 5b). SI trifid with lateral branches shorter, SII-SIII simple (Fig. 5c). Labral lamella comb-like (Fig. 5c). Premandible dark, with 2 basal and 2 inner teeth, 72–88, 74 μm long; brush well-developed (Fig. 5c). Mandible dark, apical tooth shorter than combined width of 4 inner teeth; seta subdentalis narrow reaching the base of basal inner teeth; setae interna with 7 branches, the apex of branches furcate (Fig. 5d), mandible 151–170, 161 μm long. Mentum dark, with wide bifid median tooth and 5 pairs of lateral teeth, median teeth sit much lower and are much smaller than 1 st lateral teeth; ventromental plate long running parallel to the lateral edge of mentum, reaching well beyond the base of last lateral tooth; seta submenti just posteriad to mentum aligned with 3 rd lateral tooth (Fig. 5e); mentum 94–116, 106 μm long and 110–126, 117 μm wide, ventromental plate 68–77, 74 μm long. Postmentum 132–157, 147 μm long. Abdomen. Posterior parapods wider than long, bearing around 15 simple dark claws, posterior parapod 157– 221, 174 μm long and 1166–239, 200 μm wide (Fig. 5f). Procercus almost as long as wide, bearing 5 apical setae, procercus 17–27, 21 μm long and 18–29, 24 μm wide, apical setae 107–118, 110 μm long. Four wide and conical anal tubules are present, anal tubules 177–234, 205 μm long. Diagnostic characters. The larva of Metriocnemus sp. “ Oregon ” can be separated from other related species by the combination of the following characteristics: Antenna short, 1 st antennal segment L/ W 1.2, ring organ located at mid-section of 1 st antennal segment, AR 1.7–1.8, blade subequal to flagellum; SI trifid with mid-branch longer; premandible with a well-developed brush; mentum dark, with wide bifid median tooth sitting much lower and are much smaller than 1 st lateral teeth, ventromental plate reaching well beyond the base of last lateral tooth, seta submenti aligned with 3 rd lateral tooth; posterior parapods wider than long, anal tubules semicircular. Biological notes. Larvae were collected along with leaf mines and larvae of Metriocnemus erythranthei on Claytonia sibirica, Myosotis scorpioides, and Petasites frigidus. The larvae in these samples were not observed closely, but on the Petasites, which was in the spray zone of a dripping rock seep, MWP noted that the dominant species had a dark band in the thoracic segments of the body, evidently referring to Metriocnemus sp. “ Oregon.” On this host, the mines were rather short, and the only larvae seen were window-feeding on the upper leaf surface (i.e., feeding externally and leaving the lower epidermis intact). Only leaves with mines were collected on the other two hosts, but these leaves also included larvae wandering externally. Thus, the degree to which M. sp. “ Oregon ” is a leafminer requires further investigation, and if it does feed in mines, it may be that it only does so within those initiated by M. erythranthei. Along with M. erythranthei and M. eurynotus, the Petasites collection also included two other chironomids: one larva of Boreochlus persimilis (Johannsen, 1926) (Podonominae) and one pupa of Orthocladius (Eudactylocladius) dubitatus Johannsen, 1942 (the adult female apparently emerged after the pupa was placed in ethanol). Boreochlus larvae have been previously reported to inhabit mosses in springs and small streams (Epler 2001), and Sublette et al. (1998) stated that those of O. dubitatus are probably madicolous (inhabiting thin films or sheets of water in streams, on vertical rock faces, etc.)., Published as part of Eiseman, Charles S., Namayandeh, Armin, Linden, John Van Der & Palmer, Michael W., 2023, Metriocnemus erythranthei sp. nov. and Limnophyes viribus sp. nov. (Diptera: Chironomidae: Orthocladiinae): leafminers of monkeyflowers, speedwells, and other herbaceous plants, with new observations on the ecology and habitats of other leaf-mining Chironomidae, pp. 41-68 in Zootaxa 5249 (1) on page 54, DOI: 10.11646/zootaxa.5249.1.3, http://zenodo.org/record/7685232, {"references":["Epler, J. H. (2001) Identification manual for the larval Chironomidae (Diptera) of North and South Carolina. A guide to the taxonomy of the midges of the southeastern United States including Florida. North Carolina Department of Environment and Natural Resources Division of Water Quality, St. Johns River Water Management District, Palatka, Florida, iv + 530 pp.","Sublette, J. E., Stevens, L. E. & Shannon, J. P. (1998) Chironomidae (Diptera) of the Colorado River, Grand Canyon, Arizona, USA, I: systematics and ecology. The Great Basin Naturalist, 58, 97 - 146. https: // doi. org / 10.5962 / bhl. part. 12137"]}

Chironomus sp. Material examined. USA: OREGON: Lane Co., Blue River, 44.1541, -122.324, 5.iv.2022, M.W. Palmer, extracted from leaves of Sparganium emersum (3 larvae, ANC). Biological note. The larvae were found mining in submerged portions of leaves of Sparganium emersum Rehmann (Typhaceae) in a beaver pond. The mines were only externally visible in transmitted light., Published as part of Eiseman, Charles S., Namayandeh, Armin, Linden, John Van Der & Palmer, Michael W., 2023, Metriocnemus erythranthei sp. nov. and Limnophyes viribus sp. nov. (Diptera: Chironomidae: Orthocladiinae): leafminers of monkeyflowers, speedwells, and other herbaceous plants, with new observations on the ecology and habitats of other leaf-mining Chironomidae, pp. 41-68 in Zootaxa 5249 (1) on page 64, DOI: 10.11646/zootaxa.5249.1.3, http://zenodo.org/record/7685232

Metriocnemus erythranthei Namayandeh, Eiseman, van der Linden & Palmer sp. nov. (Figs. 1–5) LSID: urn:lsid:zoobank.org:act: 524CFBAA-C5C4-48B6-A219-A71614991FB8 Holotype. USA: OKLAHOMA: Woodward Co., Boiling Spring State Park; 2.v.2021, em. 10–11.v.2021, leg. E. LoPresti and K. Toll, ex Erythranthe glabrata (1Ô, USNM). Paratypes. Canada: BRITISH COLUMBIA: Cowichan Valley, 48.837266, -123.590338, 25.iii.2022, leg. F. McGhee, ex Erythranthe guttata (1 pupa, 1 larva, USNM); USA: CALIFORNIA: Sonoma Co., Bodega Bay, Campbell Cove; 31.v.2019, em. by 3.vi.2019, leg. K. Toll, ex Erythranthe guttata (1 larva, USNM); IOWA: Winneshiek Co., Decorah, Van Peenan Spring at Van Peenan Park, 43.312834, -91.776010, 17.v.2022, em. 22.v.2022, leg. J. van der Linden, ex Veronica sp. (1Ô, USNM); OKLAHOMA: same data as holotype (1Ô, 2♀, 2 pupae, 1 larva, USNM); OREGON: Lane Co., 44.235804, -122.85404, 9.v.2022, em. 14.v.2022, leg. J. Ward, ex Claytonia sibirica (1♀, 1 pupa, USNM); Lane Co., Blue River, 44.1535, -122.328, 3.vi.2022, leg. M. W. Palmer, ex Claytonia sibirica (1Ô, 1 pupa, 1 larva, USNM); same but ex Myosotis scorpioides (1 pupa, USNM); same but ex Petasites frigidus, adults em. 12–20.vi.2022 (3ÔÔ, 2♀♀, 1 pupa, 1 larva, USNM); PENNSYLVANIA: Berks Co., Blandon, 40.461258, -75.88125, 8.v.2022, preserved 14.v.2022, leg. C. Smith, ex Veronica anagallis-aquatica (1 larva, USNM). Other material examined. USA: CALIFORNIA: Santa Cruz Co., Bonny Doon, 31.x.2012; leg. C.S. Eiseman, ex Erythranthe moschata (1 larva, JHEC); IOWA: Winneshiek Co., Decorah, Twin Springs Park, 43.297623, - 91.815370, 4.iv.2022, em. 6.iv.2022, J. van der Linden, ex Veronica sp. (1Ô, 1 pupa, 1 larva, ANC); same but 6.iv.2022 (2 pupae, 3 larvae, ANC); same location, 17.v.2022, em. 22.v.2022, J. van der Linden, ex Veronica sp. (4ÔÔ, 4 pupae, 1 larva, ANC); OREGON: Lane Co., Blue River, 44.1535, -122.328, 6.iv.2022, leg. M. W. Palmer, ex Erythranthe guttata complex (6 pupae, 12 larvae, ANC); same collection, em. 10–14.iv.2022 (3ÔÔ, 1♀, ANC); same but 28.iv.2022 (16 larvae, ANC); same but 3.vi.2022, ex Stachys cooleyae (8 larvae, ANC); same but 3.vi.2022, adult em. ~ 8.vi.2022, ex Mentha × piperita ssp. citrata (1♀, 1 pupa, ANC); same but 3.vi.2022, adult em. ~ 8.vi.2022, ex Myosotis scorpioides (1Ô, 1 larva, ANC); same but 3.vi.2022, adults em. 12–20.vi.2022, ex Petasites frigidus (1Ô, 11♀♀, 6 pupae, 4 larvae, ANC); Blue River, 44.1507, -122.324, 28.iv.2022, leg. M. W. Palmer, ex Veronica americana (11 larvae, ANC); PENNSYLVANIA: Berks Co., Blandon, 40.461258, -75.88125, 8.v.2022, preserved 14.v.2022, leg. C. Smith, ex Veronica anagallis-aquatica (1 larva, ANC). Photographed leaf mines. Canada: BRITISH COLUMBIA: Capital Regional District, Langford, Goldstream Provincial Park, 48.483147, -123.551983, 13.viii.2022, L. Ragan, Claytonia sibirica (iNat 130692120); Cowichan Valley, 48.837266, -123.590338, 25.iii.2022, F. McGhee, Erythranthe guttata (iNat 110361786); USA: ALASKA: Sitka Co., Sitka, 57.08361, -135.282176, 15.vii.2021, J. Goff, Erythranthe guttata (iNat 87446898); Sitka Co., 57.123319, -135.313118, 4.viii.2022, M. Goff, Erythranthe guttata (iNat 129592195); CALIFORNIA: Marin Co., Golden Gate National Recreation Area, 37.830228, -122.505225, 12.vi.2022, C. Chang, Erythranthe Sect. Simiolus (iNat 121533464); Napa Co., McLaughlin Preserve, 38.856449, -122.402827, 21.vi.2016, E. LoPresti, Erythranthe guttata (iNat 69586931); Santa Clara Co., San Jose, 37.13310, -121.778717, 29.iv.2022, M. Vonshak, Veronica (iNat 115791054); Sonoma Co., Sebastopol, Morelli Ln, 38.430297, -122.95137, 8.ii.2022, K. Toll, Erythranthe guttata (iNat 106618091); OREGON: Lane Co., 44.153482, -122.329189, 27.iii.2021, M. Palmer, Erythranthe?nasuta (guttata complex) (iNat 109961926); 43.942162, -123.892629, 7.v.2022, J. Ward, Claytonia cf. sibirica (iNat 116020762); 43.637107, -122.617126, 13.vii.2022, J. Ward, Claytonia sibirica (iNat 126161890); Lincoln Co., Cape Perpetua Overlook, 44.287479, -124.110256, 25.vi.2022, J. Ward, Claytonia sibirica (larvae observed, but only empty mines in photos) (iNat 123640622); Linn Co., 44.378202, -122.000636, 22.ix.2022, J. Ward, Erythranthe moschata (iNat 136120938); Marion Co., 44.883958, -122.619387, 7.vii.2022, J. Ward, Claytonia sibirica (empty mines) (iNat 125263360); PENNSYLVANIA: Berks Co., Peters Creek Spring, 27.x.2021, C. Smith, Veronica (iNat 100832107, 100832147, 100832157, 100832185); WASHINGTON: King Co., Vashon, 47.390545, -122.489901, 24.iv.2020, H. Parker, Myosotis (iNat 44625412); same but 3.v.2020 (iNat 52704648, 59353928, 59355328); WISCONSIN: Waukesha Co., Eagle Spring and Fen, 42.924285, -88.468514, 24.ix.2014, D. Carter, Erythranthe geyeri (iNat 893113). Etymology. The new species is named after Erythranthe (monkeyflowers), one of the plant genera commonly inhabited by the larvae. Description. Male (n = 11). Total length 1.9–2.5, 2.2 mm. Wing 1.8–1.9 mm long and 0.45 mm wide. Coloration. Head, thorax, legs, tergites, sternites II–VIII, and hypopygium dark brown. Wings, halters, and sternite I greyish. Head. Antenna with 12 flagellomeres, last flagellomere with 8 sensilla chaetica, second to third segments each with 2 sensilla chaetica, groove starts at third segment, AR 0.9. Eyes bare, with wedge-shaped dorsomedial extension. Temporal setae 32 in several rows. Tentorium with bulging apex, 183–194, 189 μm long (Fig. 1a). Clypeus rectangular, 83–96, 89 μm long and 121–154, 138 μm wide, bearing 26 setae, setae 73–88, 81 μm long. Palpal segment lengths (in μm): 72–76, 74; 51; 129–159, 144; 80–95, 88; 141–167, 154. Third palpomere with 3 sensilla clavata. Thorax (Fig. 1b). Acrostichals 25–30, 27; dorsocentrals 79–109, 94 in multiple rows; prealars 21–24, 22; scutellars 42–44, 43 in three rows; supraalars 2–3. Antepronotal lobes developed, with a gap, 14–16, 15 lateral setae. Wing (Fig. 1c). Brachiolum with 10 setae. Squama with 18 setae. Sc with 30–33, 32; R with 32–42, 37 and R 1 with 35–43, 39 setae, R 4+5 with 50–54, 52 setae, and M with 25–28, 27 setae, other veins bare. Costa well-extended, extension 77 μm. R 4+5 ends just above M 3+4. Anal lobe not projecting (Fig. 1c). Legs. Tibia of all legs with long sparse beard; hind and mid femur with keel. Pulvilli very small. Fore tibia spur 62–72, 67 μm long, mid tibia spurs 34–39, 36 and 33–34 μm long, hind tibia spurs 55–65, 60 and 28 μm long, hind tibia comb with around 12 spines. 2 pseudospurs on ta 1 of mid and hind legs. Lengths and proportions of legs as in Table 1. Hypopygium (Fig. 1d). Tergite IX with around 14 long setae close to the base of anal point. Anal point short, narrowly triangular, apex rounded; anal point 32–46, 39 μm long and 17–23, 20 μm wide. Virga present, consisting of about 8–9 spines, 38–46, 42 μm long. Sternapodeme nearly straight, 124–136, 130 μm long. Phallapodeme 89–95, 92 μm long. Inferior volsella located anteriorly on gonocoxite, slightly bulging, covered in numerous simple setae. Gonostylus 113–121, 117 μm; crista dorsalis a preapical short triangle. Gonocoxite 231–246, 238 μm long. HR 2, HV 2.5–2.9, 2.7. Female (n = 5). Total length 2.8–3.1, 3.0 mm. Wing 1.8–1.9 mm long and 0.6 mm wide. Coloration. Humeral, anepisternal, preepisternum, and posnotum light brown, remainder same as the male. Head. Antenna with 5 flagellomeres, last flagellomere 60–73, 67 μm long; last flagellomere with 12 sensilla chaetica, 1 st –4 th segments each with 2 sensilla chaetica, AR 0.3 (Fig. 2a). Eyes bare, with short wedge-shaped dorsomedial extension. Temporal setae 43–50, 47 in several rows. Tentorium with bulging apex, 192–198, 195 μm long. Clypeus rectangular, 101–107, 104 μm long and 149–165, 157 μm wide, bearing 30–36, 33 setae, setae 79–100, 81 μm long. Palpal segment lengths (in μm): 61; 36–42, 39; 122–156, 139; 98–118, 108; 135–181, 158. Third palpomere with 1 sensilla clavata. Thorax. Acrostichals 34–40, 37; dorsocentrals 105–112, 109 in multiple rows; prealars 27–33, 30; scutellars 50– 54, 52 in two rows; supraalars 5.Antepronotal lobes developed, with a gap, 14–17, 15 lateral setae. Humeral pit small. Wing (Fig. 2b). Brachiolum with 9 setae. Squama with 19–22, 22 setae. R with 40–57, 50; R 1 with 27–40, 34 setae; R 4+5 with 50 setae, and M with 24–25 setae, other veins without setae. Costa well-extended, extension 45 μm. R 4+5 ends just above M 3+4. Anal lobe not projecting Legs. Hind-fore tibia with long sparse beard, hind and mid femur with keel. Pulvilli very small. Fore tibia spur 37–41, 39 μm long, mid tibia spurs 31–38, 34 and 30 μm long, hind tibia spurs 45–56, 51 and 25–33, 29 μm long, hind tibia comb with around 15 spines. 2 pseudospurs on ta 1 of mid and hind leg. Lengths and proportions of legs as in Table 2. Genitalia (Figs. 2c–d). Seminal capsules small, semi-circular, spermathecal ducts without loops, with small bulb (Fig. 2c), seminal capsule 67–68 µm long and 54–67, 61 µm wide. Notum 228–271, 249 µm long. Gonapophysis VIII divided into large ventrolateral and smaller dorsomesal lobe (Fig. 2c). Apodeme lobe distinct. Gonocoxite developed with around 24 setae (Fig. 2d). Tergite IX undivided (Fig. 2d). Cercus pediform, 152–156, 154 µm long, and 60 µm wide. Pupa (n = 8). Total length 4.5 mm. Coloration: Head and thorax brown. Abdomen golden. Cephalothorax. Frontal setae absent, frontal apotome rugose (Fig. 3a). Antennal sheet without pearls or spines above to pedicels. Thorax rugose, horn absent. Wing sheet nearly smooth, 1.1–1.2 mm long and 0.32–0.39, 0.35 mm wide. Abdomen (Figs. 3b–c). Tergite I with no posterior row of tubercles and no shagreens. Tergite II with no shagreens, a single row of posterior tubercles; tergites III–VIII with a single row of posterior tubercles; tergites III–VI with anterior crescent-shaped shagreens, becoming more prominent towards segment VI; tergites VII–VIII with shagreens more prominent anteriorly, becoming reduced from mid to posterior regions; tergite IX covered in shagreen (Fig. 3b). Sternites I, II, and VIII bare; sternites III–VII with posterior shagreens; sternite IX bare (Fig. 3c). Anal lobe 233–278, 256 µm long and 159–178, 171 µm wide; with two to three extremely short and hairlike macrosetae, 4.3–4.7, 4.5 µm long. Genital sac shorter than anal lobes, 163–167, 165 µm long and 105–122, 113 µm wide. Larva (n = 4). Total length 4.4 mm. Head 327–407, 379 μm long, 234–303, 268 μm wide. Coloration (Fig. 3d). Head capsule ventrally light brown, dorsally dark brown. Occipital margin much darker in contrast to the remainder of the head. Abdomen yellowish green. Head. Head L/ W 1.3 –1.5, 1.4. Antenna short, 5 segmented; 1 st antennal segment 1.5–2 × as long as wide; ring organ located at basal ¼ of 1 st segment; AR 1.3–1.5, 1.4, blade longer than the flagellum, blade 25–27, 26 μm long, B 2 6 μm long; antennal segments length in μm: 28–32, 8, 3.4–4, 3, 6–6.6 (Fig. 3e). SI divided into 4 branches, SIISIII simple (Fig. 3f). Premandible dark, with 2 basal and 2 inner teeth, 68–76, 72 μm long, brush developed (Fig. 3f). Mandible dark, apical tooth shorter than combined width of 4 inner teeth; seta subdentalis narrow reaching the base of basal inner teeth; setae interna with 7 branches, the apex of branches furcate (Fig. 3g), mandible 137–149, 141 μm long. Mentum dark, with wide bifid median tooth and 4 pairs of lateral teeth, ventromental plate running parallel to the lateral edge of mentum, reaching the base of last lateral tooth; seta submenti just posteriad to mentum aligned with 2 nd lateral teeth (Fig. 3h); mentum 86–98, 93 μm long and 105–142, 120 μm wide, ventromental plate 47–48 μm long and 10 μm wide. Abdomen. Posterior parapods well-reduced, much wider than long, bearing around 15 simple claws, posterior parapod 62 μm long and 140 μm wide. Procercus almost as long as wide, bearing 5 apical setae, procercus 19–20 μm long and 22–23 μm wide, apical setae 92–105, 98 μm long. Diagnostic characters. Metriocnemus erythranthei can be separated from other related species by the combination of the following characteristics: Adult male with AR 0.9; temporal setae 32 in several rows; tentorium with bulging apex; anal point short, moderately wide, and triangular, apex slightly rounded; virga consists of about 8–9 spines; inferior volsella located anteriorly and slightly bulging; crista dorsalis of gonostylus preapical, short and triangular. Adult female with AR 0.9; last flagellomere 67 μm long, last flagellomere/2 nd flagellomere 1.2; temporal setae 43–50 in several rows; seminal capsules small, semi-circular, spermathecal ducts without loops, with small bulb; gonapophysis VIII divided into large ventrolateral and dorsomesal lobe; apodeme lobe distinct; gonocoxite well-developed with around 24 setae; cercus pediform. Pupa with frontal setae absent; frontal apotome rugose; tergites I–II with no shagreens; tergites III–VIII with shagreens; tergites II–VIII with a single row of posterior tubercles. Sternites I, VII, and VIII bare; sternites III–VI with posterior shagreens; sternite IX covered in shagreen. Anal lobe with two to three extremely short and hairlike macrosetae; genital sac shorter than anal lobes. Larva with short antenna; basal antennal segment 1.5–2 × as long as wide; AR 1.4, blade longer than the flagellum; SI divided into 4 branches; premandible with 2 basal, 2 inner teeth and brush; mandible apical tooth shorter than combined width of 4 inner teeth; mentum with wide bifid median tooth and 4 pairs of lateral teeth; posterior parapods wellreduced. Biological notes (Figs. 4–5). Metriocnemus erythranthei mines leaves of at least three different monkeyflower species: Erythranthe glabrata (Kunth) G.L.Nesom in Oklahoma (Fig. 4a), E. guttata (DC.) G.L.Nesom (or E. guttata complex) in British Columbia, Oregon, and California (Fig. 4b), and E. moschata (Douglas ex Lindl.) G.L.Nesom in Oregon and California (Figs. 4c–d). Mines, probably representing this species, have been photographed in Alaska on E. guttata, as well as in Wisconsin on E. geyeri (Torr.) G.L.Nesom. In Oregon, Iowa, and Pennsylvania, M. erythranthei mines leaves of Veronica americana (Raf.) Schwein. ex Benth. and V. anagallis-aquatica L. (Plantaginaceae) (Figs. 4e–g, 5a–b), and in Oregon it has also been found to feed on Mentha aquatica L. and Stachys chamissonis var. cooleyae (A.Heller) G.A.Mulligan & D.B.Munro (Lamiaceae), all of which, like Erythranthe, belong to the order Lamiales. In Oregon, confirmed hosts also include plants in three additional orders— Asterales (Asteraceae: Petasites frigidus (L.) Fr.), Boraginales (Boraginaceae: Myosotis scorpioides L.), and Caryophyllales (Montiaceae: Claytonia sibirica L.). Larvae and mines have also been photographed on Myosotis in Washington and on Claytonia in British Columbia. Leaf-mining chironomids representing this or a similar species have been found on still other plants in the Pacific Northwest; see the final section of this paper for details. The leaf mines of M. erythranthei are essentially linear throughout, initially narrow and gradually widening, sometimes becoming somewhat irregular or blotchy toward the end. They are mostly full-depth (all of the mesophyll being consumed, leaving only the leaf epidermises intact), with frass in roughly oval pellets that are scattered at random (Figs. 4a, b, d, f). The larvae are able to exit their mines and establish new ones (Figs. 4c, e, g). In a thorough review of the literature on North American leaf-mining insects (Eiseman 2022), we found no records of any leafminer occurring on Erythranthe in nature. The only published record involving this host genus is that of Oatman (1959), who conducted greenhouse experiments with the polyphagous fly Liriomyza sativae Blanchard, 1938 (Agromyzidae). He observed mining in E. guttata; a few larvae survived to the point of pupating, but no adults emerged. Were Liriomyza mines to occur on Erythranthe in nature, they would be easily recognizable by the characteristic pattern of frass in strips on alternating sides of the channel. The only previously known leafminer of Veronica spp. in North America is another agromyzid, Phytomyza crassiseta Zetterstedt, 1860. The formation of the puparium (as opposed to a naked pupa) within the leaf is the most obvious feature distinguishing mines of P. crassiseta from those of M. erythranthei. Claytonia likewise has a single recorded leafminer, the Caryophyllales generalist Pegomya flavifrons (Walker, 1849) (Anthomyiidae). The persistent white eggshell at the beginning and the distinctly blotchy ultimate shape will distinguish mines of P. flavifrons from those of M. erythranthei. The other confirmed hosts of M. erythranthei are also mined by various agromyzid species, but all of these produce mines that are more or less confined to one leaf surface and differ in shape and frass pattern (Eiseman 2022). In the three leaf-mining Cricotopus species, pupation occurs within the mine, but when the adult is ready to emerge, the pupa breaks out of the mine and swims to the water surface (Berg 1950). The habits of M. erythranthei are generally similar. On Erythranthe in British Columbia and on Veronica in Iowa, mines have been collected with pupae inside, each surrounded by a gelatinous mass (Figs. 5a–c, e). In collections of larvae from Claytonia and Erythranthe elsewhere, pupation has taken place outside the mines (at least sometimes in a gelatinous mass), but the leaves had more or less deteriorated by this point. Based on our observations of M. erythranthei on Veronica, the pupa remains in the mine until shortly before emergence of the adult. In one instance the pupal exuviae were found protruding from the mine, with only the tip of the abdomen still inside (Fig. 5d). In another, the exuviae were found loosely adhering to plant material seve, Published as part of Eiseman, Charles S., Namayandeh, Armin, Linden, John Van Der & Palmer, Michael W., 2023, Metriocnemus erythranthei sp. nov. and Limnophyes viribus sp. nov. (Diptera: Chironomidae: Orthocladiinae): leafminers of monkeyflowers, speedwells, and other herbaceous plants, with new observations on the ecology and habitats of other leaf-mining Chironomidae, pp. 41-68 in Zootaxa 5249 (1) on pages 43-51, DOI: 10.11646/zootaxa.5249.1.3, http://zenodo.org/record/7685232, {"references":["Eiseman, C. (2022) Leafminers of North America. Privately published e-book, clxvii + 2213 pp. Available from: http: // charleyeiseman. com / leafminers (accessed 9 January 2023)","Oatman, E. R. (1959) Host range studies of the melon leaf miner, Liriomyza pictella (Thomson) (Diptera: Agromyzidae). Annals of the Entomological Society of America, 52, 739 - 741. https: // doi. org / 10.1093 / aesa / 52.6.739","Berg, C. O. (1950) Biology of certain Chironomidae reared from Potamogeton. Ecological Monographs, 20, 83 - 101. https: // doi. org / 10.2307 / 1943546"]}

Limnophyes spp. (Fig. 7b) Material examined. USA: IOWA: Winneshiek Co., Twin Springs Park, 11.vi.2017, em. ~ 21.vi.2017, J. van der Linden, ex Veronica anagallis-aquatica (2♀); same data but collected 29.i.2018 (1 larva). These specimens were examined by A. Namayandeh in 2018 and their current location is unknown. Biological notes. Each of the collections listed above consisted of a few Veronica stems with attached leaves in which chironomid larvae were forming linear mines. The only larva photographed from the first collection was consistent with M. erythranthei. The two female Limnophyes adults emerged about ten days after the first collection. One of these was light reddish-brown and was identified as Limnophyes cf. carolinensis Saether, 1975 (https:// bugguide.net/node/view/1396485). The other was grayish-black and appeared to be L. viribus (https://bugguide. net/node/view/1396495). The only other insect to emerge from this collection (about a day earlier) was an adult of Scaptomyza pallida (Zetterstedt, 1847) (Drosophilidae), a saprophagous species that sometimes develops as an inquiline in mines made by other fly larvae, including Zygoneura calthella Eiseman, Heller & Rulik, 2016 (Sciaridae) on marsh marigold (Ranunculaceae: Caltha palustris L.) (Eiseman et al. 2016) and Agromyza parvicornis Loew, 1869 (Agromyzidae) on corn (Poaceae: Zea mays L.) (C. Eiseman, unpublished). Whether the Limnophyes adults arose from the observed leaf-mining larvae or were themselves inquilines or contaminants was not known with certainty. About two weeks after the second collection of Veronica leaf mines (on 12 February 2018), actively wriggling Metriocnemus pupae were observed in the leaf mines, and in one case, in a stem. On the same date, an adult male of another orthocladiine, Corynoneura lobata Edwards, 1924, was found dead in the rearing container; its pupal exuviae were not located and the larval habits of this individual are unknown. Corynoneura lobata has an aquatic larva inhabiting mainly fast-flowing waters (Cranston 1982). Also on this date, the now preserved Limnophyes larva, which was initially identified as L. cf. carolinensis but appears consistent with L. viribus, was observed moving within an existing linear mine that was substantially wider than its body, consuming small patches of mesophyll here and there along the margins (Fig. 12b; video: https://youtu.be/0h3Bvc0DfgE). The following day, the first adult Metriocnemus emerged from one of the pupae in the mines. Based on the available evidence, we believe that the Limnophyes species we have reared do not establish their own mines but feed as inquilines in mines created by Metriocnemus larvae., Published as part of Eiseman, Charles S., Namayandeh, Armin, Linden, John Van Der & Palmer, Michael W., 2023, Metriocnemus erythranthei sp. nov. and Limnophyes viribus sp. nov. (Diptera: Chironomidae: Orthocladiinae): leafminers of monkeyflowers, speedwells, and other herbaceous plants, with new observations on the ecology and habitats of other leaf-mining Chironomidae, pp. 41-68 in Zootaxa 5249 (1) on pages 61-62, DOI: 10.11646/zootaxa.5249.1.3, http://zenodo.org/record/7685232, {"references":["Eiseman, C. S., Heller, K. & Rulik, B. (2016) A new leaf-mining dark-winged fungus gnat (Diptera: Sciaridae), with notes on other insect associates of marsh marigold (Ranunculaceae: Caltha palustris L.). Proceedings of the Entomological Society of Washington, 118, 519 - 532. https: // doi. org / 10.4289 / 0013 - 8797.118.4.519","Cranston, P. S. (1982) A key to the larvae of the British Orthocladiinae (Chironomidae). Freshwater biological association scientific publication, 45, 1 - 152."]}

Paraphaenocladius exagitans exagitans (Johannsen, 1905) and Paraphaenocladius impensus impensus (Walker, 1856) (Fig. 13) Material examined. USA: IOWA: Allamakee Co., Yellow River State Forest, 43.174434, -91.221148, 13.x.2017, em. 23.xi.2017, J. van der Linden, ex Marchantia polymorpha (1♀, P. exagitans exagitans, ANC); same but em. 2.xii.2017 (1♀, P. exagitans exagitans, USNM); same but em. 1.i.2018 (1♀, P. impensus impensus, USNM); same but em. 15.i.2018 (1Ô, P. exagitans exagitans, ANC); same but em. 29.i.2018 (1♀, P. impensus impensus, ANC); same but em. 12.ii.2018 (1♀, P. exagitans exagitans, USNM); OREGON: Lane Co., Blue River, 44.1535, -122.328, 28.iv.2022, em. 29.iv–7.v.2022, leg. M. W. Palmer, ex Marchantia sp. (2ÔÔ, 1♀, 1 larva, P. exagitans exagitans, USNM; 1Ô, P. exagitans exagitans, ANC). Biological notes. The nominate subspecies of P. exagitans is known from Ontario and throughout the USA as well as China and Japan. It has previously been reared from moist soil at the margins of rivers, streams, seepage, and springs, but none of the published label data mention herbivory (Saether & Wang 1995). Paraphaenocladius impensus s. str. is recorded from Manitoba, Minnesota, Greenland, and northern Europe, where it has been found in moist soil at the margins of lakes, springs, meadows, and alder carr; there have likewise been no observations of herbivory in this species (Saether & Wang 1995). Nematoceran larvae were found mining in thalli of the liverwort Marchantia polymorpha L. (Marchantiaceae) in Iowa in mid-October 2017. The habitat was a ditch in which the soil varied from saturated to submerged in several centimeters of standing water. The larvae initially formed digitate mines with short radiating galleries, and later formed brown blotches. Unlike Metriocnemus mines, which have frass scattered throughout, these mines were mostly clean with frass accumulating around the rim of the entry hole. The first adult of P. exagitans emerged on 23 November from an oblong, dark brown cocoon, composed of frass, which was formed on the surface of a thallus (Figs. 13a, b). Its pupal exuviae were left on the surface of the cocoon. The frass appeared to have been produced by the larva feeding on the surface of the thallus. Two other adults of P. exagitans and three of P. impensus emerged over the next few months. Two larvae were observed mining in fresh thallus growth in late December. Unfortunately, none of the larvae were preserved, and we are unable to confirm that either Paraphaenocladius species feeds as a thallus miner. The photographed larvae are all Sciaridae, and although this rearing effort did not produce any adult sciarids, in 2022 JvdL reared adult sciarids from similar larvae found mining liverwort thalli at another site in Iowa. The sciarid larvae pupated inside their mines. A collection of Marchantia cf. polymorpha in Oregon in late April 2022 produced four adults and a larva of P. exagitans exagitans, along with eight adults of Boreochlus persimilis. Although some empty mines of an unknown insect were seen in a nearby colony of this plant, none were observed in the collected sample, and since the rhizoids of the collected plants were thick and encompassed a substantial amount of mucky organic matter, it is unclear what these midges were feeding on as larvae. The repeated rearing of Paraphaenocladius adults from collections of Marchantia suggests more than a casual association with this plant, but further investigation is required to determine whether the larvae feed as thallus miners, as secondary inhabitants in sciarid mines, as external feeders on the thallus surface or concealed in the rhizoids, or some combination of these., Published as part of Eiseman, Charles S., Namayandeh, Armin, Linden, John Van Der & Palmer, Michael W., 2023, Metriocnemus erythranthei sp. nov. and Limnophyes viribus sp. nov. (Diptera: Chironomidae: Orthocladiinae): leafminers of monkeyflowers, speedwells, and other herbaceous plants, with new observations on the ecology and habitats of other leaf-mining Chironomidae, pp. 41-68 in Zootaxa 5249 (1) on pages 62-64, DOI: 10.11646/zootaxa.5249.1.3, http://zenodo.org/record/7685232, {"references":["Saether, O. A. & Wang, X. (1995) Revision of the genus Paraphaenocladius Thienemann, 1924 of the world (Diptera: Chironomidae, Orthocladiinae). Entomologica scandinavica Supplement, 48, 3 - 69."]}

Expanded analysis and results

Supplemental material

Background: To address multidrug resistance, we developed engineered Cationic Antimicrobial Peptides (eCAPs). Lead eCAP WLBU2 displays potent activity against drug-resistant bacteria and effectively treats lethal bacterial infections in mice, reducing bacterial loads to undetectable levels in diverse organs. Objective: To support development of WLBU2, we conducted a mass balance study. Methods: CD1 mice were administered 10, 15, 20 and 30 mg/kg of QDx5 WLBU2 or a single dose of [14C]-WLBU2 at 15 mg/kg IV. Tolerability, tissue distribution and excretion were evaluated with liquid scintillation and HPLC-radiochromatography. Results: The maximum tolerated dose of WLBU2 is 20 mg/kg IV. We could account for greater than >96% of the radioactivity distributed within mouse tissues at 5 and 15 min. By 24h, only ~40-50% of radioactivity remained in the mice. The greatest % of the dose was present in liver, accounting for ~35% of radioactivity at 5 and 15 min, and ~ 8% of radioactivity remained at 24h. High radioactivity was also present in kidneys, plasma, red blood cells and lungs, while less than 0.2% of radioactivity was present in brain, fat, or skeletal muscle. Urinary and fecal excretion accounted for 12.5 and 2.2% of radioactivity at 24h. Conclusion: WLBU2 distributes widely to mouse tissues and is rapidly cleared with a terminal radioactivity half-life of 22 h, a clearance of 27.4 mL/h/kg, and a distribution volume of 0.94 L/kg. At 2-100 μg-eq/g, the concentrations of 14C-WLBU2 appear high enough in the tissues to account for the inhibition of microbial growth.

To evaluate the effect of demographic disparities on language outcomes in a diverse group of children who are deaf or hard of hearing.Retrospective cohort study.UCSF Benioff Children's Hospital (a tertiary care center).Forty-four patients aged18 years were identified with sensorineural hearing loss managed with a behind-the-ear hearing aid or cochlear implant. Demographic and clinical data were extracted from the medical record. The primary outcome measure was the Preschool Language Scales-5 at least 6 months after intervention. Predictors of language outcome were assessed: hearing level at the time of hearing intervention, cochlear implant status, age of identification and intervention, travel time to site of hearing care, home language, race/ethnicity, insurance type, and Access Challenge Index-a novel measure of educational environment and family support based on the Child Cochlear Implant Profile. Multivariate and univariate analysis assessed predictors for association with intervention and receptive, expressive, and total language scores.Overall 82% of patients had cochlear implants. The median age at hearing intervention was 12 months. The sample was 59% female, 52% non-White, and 61% publicly insured, and 20% had a non-English primary home language. Accounting for multiple demographic and clinical predictors, a high Access Challenge Index score was independently associated with longer time to intervention (Access Challenge Index-a novel comprehensive measure of educational and family environment-is a strong independent predictor of language outcomes in children who are deaf or hard of hearing.