2009 NORTHEASTERN NATURALIST 16(2):285–306 Biology of the Caddisfly Oligostomis ocelligera (Trichoptera: Phryganeidae) Inhabiting Acidic Mine Drainage in Pennsylvania Lori A. Redell1,*, Wayne K. Gall2, Robert M. Ross1, and David S. Dropkin1 Abstract - Oligostomis ocelligera (a phryganeid caddisfly) is reported for the first time from a degraded lotic system—a first-order stream in north-central Pennsylvania that was severely impacted by acid mine drainage. Although uncommonly collected and poorly known, O. ocelligera maintained a substantial population in the mine discharge, free of competition from Plecoptera, Ephemeroptera, and other species of Trichoptera. It thrived under conditions of very low pH (2.58–3.13), high concentrations of sulfate (542 mg/L) and heavy metals (Fe 12 mg/L, Mn 14 mg/L, Al 16 mg/L), and a nearly uniform springbrook-like temperature regime. More than 350 larvae were collected from deposits of leaves and woody detritus in a pool 0.32 km downstream from the mine entrance over a two-year period. Measurement of headcapsule widths yielded a multimodal distribution with five peaks, corresponding to five instars, in conformity with Dyar’s Law. Eighty-three egg masses were observed along the stream channel from 3 June to 12 November at a mean distance of 6.1 cm above the water surface in moist, protected locations such as under moss mats or in crevices of logs. Eggs began hatching by mid-summer, first-instar larvae were present in samples from August–October, all five instars were represented in October, instars II–V were still present in December, but only instars IV and V were represented in samples collected from March to July. The extended periods of oviposition and larval recruitment, together with a remarkably protracted flight period of six months (29 April–30 October), led to the conclusion that the population of O. ocelligera at the mine site exhibited an asynchronous univoltine life cycle. Measurement of the width of the anterior border of the frontoclypeal apotome confirmed Wiggins’ proposal that this metric is useful for distinguishing final instar larvae of O. ocelligera from its only Nearctic congener, O. pardalis. Occupied pupal cases were found embedded in sodden logs from 8 April to 10 June. Pupae had mandibles reduced to membranous lobes. A silken mesh closing the anterior end case of the pupal case is reported for the first time in O. ocelligera, representing the third evolutionary reversal for this behavioral character in the phylogeny of phryganeid genera proposed by Wiggins. Adults exhibited only diurnal flight, and were absent from light traps deployed on five nights. Females displayed more cryptic behavior, and their wing pattern was distinctly duller in color than males. Introduction Knowledge of the life history and biology of the phryganeid caddisfly, Oligostomis ocelligera (Walker) (Trichoptera: Phryganeidae), has advanced 1US Geological Survey, Leetown Science Center, Northern Appalachian Research Branch, 176 Straight Run Road, Wellsboro, PA 16901. 2New York State Department of Health, 584 Delaware Avenue, Room 202, Buffalo, NY 14202. *Corresponding author – lori_redell@usgs.gov. 286 Northeastern Naturalist Vol. 16, No. 2 little since its larva was first described more than eighty-five years ago (Lloyd 1921; as Neuronia stygipes). This dearth of knowledge may be attributed to the highly localized populations of O. ocelligera, the relatively uncommon collection of adults (Wiggins 1998), and the lack of diagnostic characters other than size for distinguishing the larva of O. ocelligera from its congener in eastern North America, Oligostomis pardalis (Walker) (Wiggins 1996a, 1998). The infrequent collection of adults may be an artifact of collection methods: coincident with its obligatory daytime flight (Lloyd 1921, Wiggins 1998), O. ocelligera is not known to be attracted to ultraviolet and/or mercury vapor lights like most other species of caddisflies. The relative paucity of collection records for O. ocelligera is ironic since its wing pattern is among the most brightly contrasting and strikingly colored of the Trichoptera (Ross 1944, Wiggins 1998). Oligostomis ocelligera has a relatively broad range in eastern North America, from Newfoundland to Wisconsin and south to Tennessee (Wiggins 1998). However, sparse occurrences of O. ocelligera have been reported in Tennessee (Etnier and Schuster 1979, Wiggins et al. 2001), Wisconsin (Karl and Hilsenhoff 1979, Longridge and Hilsenhoff 1973), New York (Betten 1934, Wiggins 1998), and Pennsylvania (Masteller and Flint 1992, 1998). Prior to our collection of all life-history stages of O. ocelligera from 2002–2004 in Tioga County, PA, there were only three published records for this species in Pennsylvania (Masteller and Flint 1998): Bear Run Nature Reserve in Fayette County (11 larvae; Seward 1977), a bog in Somerset County (5 larvae in pitcher plants; Hamilton et al. 1998); and Spring Creek in Warren County (1 adult male; Masteller and Flint 1998). A further impediment to elucidating the biology of O. ocelligera has been the problem of distinguishing the larva of this species from its sympatric congener, O. pardalis. Wiggins (1960b, 1996a, 1998) stated that larvae of the two North American species are separable only by size. Wiggins (1996a) suggested using the width of the anterior border of the frontoclypeal apotome of final instar larvae as an effective index to distinguish the two species, but lacked sufficient specimens of proven identity to establish a range of measurements for each species. Previously published information on the biology of O. ocelligera is limited to that provided by Lloyd (1921) and Wiggins (1996a, 1998). Lloyd (1921) reported that larvae of O. ocelligera were “not uncommon” in a short area near the headwaters of Argus Brook in the McLean Bogs Natural Area (known then as the Lloyd-Cornell Reservation) in northeastern Tompkins County, NY. He observed larvae of this species crawling actively on the stream bottom until mid- to late April when they burrowed into the soil or dead wood for pupation. Considering the genus Oligostomis as a whole, Wiggins (1996a, 1998) reported that larvae inhabit small cool streams in forests, generally in areas of slow current where leaves accumulate, and where they construct cases in ring-like sections that are composed of leaf and bark pieces. During the last two weeks of May, Lloyd found adults common 2009 L.A. Redell, W.K. Gall, R.M. Ross, and D.S. Dropkin 287 as they flew in slow, jerky flights close to low vegetation or skimmed the stream surface in daylight, but were restricted to the borders of the stream from which they had emerged. When resting, they sought shelter on low weeds or grass. Lloyd found only fragments of disintegrated leaves in the guts of larvae he examined. Although Wiggins (1996a) did not examine larvae of O. ocelligera, he reported that three larvae of O. pardalis contained animal remains in addition to filamentous algae and vascular plant tissue. Wiggins (1996a, 1998) also cited an analysis of gut contents of late instar larvae of the European species, Oligostomis reticulata (Linnaeus), in which approximately 10% of the food consisted of higher plants, while insects and other invertebrates accounted for the balance. The discovery of a relatively large population of larvae of O. ocelligera inhabiting acid mine drainage in north-central Pennsylvania provided a unique opportunity for us to study the life history, behavior, larval feeding habits, and environmental tolerance of O. ocelligera. A series of final instar larvae also allowed us to assess the value of using the width of the anterior border of the frontoclypeal apotome as a diagnostic character to distinguish larvae of the broadly sympatric species, O. ocelligera and O. pardalis, as proposed by Wiggins (1996a, 1998). Field-site Description The Anna S. Mine is located approximately 5.5 km north of Morris in Tioga County, PA (Figs. 1, 2), and drains an area of approximately 134 ha (Reed 1980). Deep mining occurred from the late 1890s into the 1940s. During 2002 and 2003, drainage from the mine was continuous and flowed down a mountainside at a rate of approximately 0.01 m3/s for 1.8 km before entering Wilson Creek. For the first 0.5 km, channel gradient was gentle (1%), Figure 1. Photograph of mine entrance. 288 Northeastern Naturalist Vol. 16, No. 2 Figure 2. Location of Anna S. Mine study site. then became abruptly steep (12%), with large boulder outcrops and braided channels. Maximum channel width was 2.6 m with a maximum depth of 18.5 cm during base-flow conditions of summer. In January 2004, about 90% of the flow from the mine (Fig. 1) was diverted to passive treatment systems, reducing channel width to 1.4 m and depth to 10.7 cm. The stream channel was devoid of vegetation, except for Klebsormidium (unbranched green filamentous alga), a few filaments of Cladophora (branched green filamentous alga), and Eunotia exigua (a diatom). Riparian vegetation consisted of Vaccinium angustifolium Ait. (Lowbush Blueberry), Sphagnum spp. (peat moss), Betula alleghaniensis Britt. (Yellow Birch), Tsuga canadensis (L.) Carr. (Eastern Hemlock), and Pinus strobus L. (Eastern White Pine). Methods Species identification Adults were identified using the key to families of North American Trichoptera in Wiggins (1996b), as well as the key to genera of adult phryganeids and the key to species of adult Oligostomis in Wiggins (1998). Genitalic morphology of adults, supplemented by forewing length, was used to differentiate this species from O. pardalis. Larvae were identified using the family key in Wiggins (1996a) and the key to genera of Phryganeidae in Wiggins (1998). Since the width of the anterior border of the frontoclypeal apotome of mature larvae 2009 L.A. Redell, W.K. Gall, R.M. Ross, and D.S. Dropkin 289 approximated that proposed by Wiggins (1996a, 1998) for O. ocelligera, we provisionally identified the larvae as this species. Species identification of larvae was subsequently confirmed by association with a long series of adult males of O. ocelligera collected along the stream discharging from the Anna S. Mine. Voucher specimens of O. ocelligera have been deposited in the Cornell University Insect Collection (CUIC), Ithaca, NY, under Lot #1258: 27 male and 3 female adults, 2 pupae (1 pharate male) with pupal cases, and 16 final instar larvae (8 larvae in cases). Sampling and laboratory analyses Water samples were taken at monthly intervals from July 2002 to August 2004 in a stream pool 0.32 km from the mine entrance where Oligostomis larvae were found. Due to the remoteness of the site and adverse weather conditions, sampling could not be conducted during January and February. Measurements of pH, conductivity, temperature, and dissolved oxygen were made in the field with YSI Model 60 and 85 meters (Yellow Springs, OH). Data for discharge and pH were compared with those obtained in 1996–98 by Hedin Environmental (2004). Canopy cover was visually estimated by noting the general proportion of open to shaded area. Larvae, pupae, and adults of O. ocelligera were also sampled from July 2002 to August 2004, with the exception of January and February. Larvae were randomly sampled with a kick net (46-cm rectangular frame, 600-micron mesh) during three 1-min periods each month at the aforementioned pool, and preserved in 70% ethanol. A drift net (25-cm diameter x 65-cm length, 500-micron mesh) was placed in a riffle 1.7 m downstream from the pool for 24 h at monthly intervals to determine the taxonomic composition of macroinvertebrates in the stream drift. Leaf packs, each filled with 5 g of leaves of Acer sp. (maple), were placed in the pool to provide artificial larval habitat where larvae were concentrated. The leaf packs were examined monthly to determine the acceptance/rejection response of O. ocelligera. Head-capsule widths of 357 preserved larvae of O. ocelligera, which were collected with a kick net in the pool during 2002–2004, were measured to the nearest 0.05 mm using an ocular micrometer in a Leica MZ6 dissecting microscope. Head-capsule widths, in intervals of 0.05 mm, were plotted against number of larvae to determine the range of head capsule widths for each instar. The number of individuals of each instar present each month was then plotted to establish a temporal distribution of larval instars. Head capsule width, total length, and the width of the anterior border of the frontoclypeal apotome of 11 larvae collected in July 2002 were measured to determine morphological characteristics of final instar larvae. The measurements of the frontoclypeal apotome were compared with those of Wiggins (1996a, 1998) to determine the effectiveness of this index in distinguishing the two North American species of Oligostomis. To assess feeding habits of late instars, guts were dissected from the 45 larvae collected in July 2002 according to the procedures outlined by 290 Northeastern Naturalist Vol. 16, No. 2 Cummins (1973). Gut contents were examined under dissecting and compound microscopes. Pupae and pupal cases were collected from submerged soft, decaying logs and preserved in 70% ethanol. Pupal density and dimensions of the logs were recorded. Ten pupal cases were dissected to recover the exuviae of terminal instar larvae. The width of the anterior border of the frontoclypeal apotome was measured for each exuvium to supplement measurements made from final instar larvae as mentioned above. Flight period was determined from adult collections that were made by sweeping riparian vegetation with a short-handled net. Sampling frequency for adult O. ocelligera was generally about 2 h, two to three times per week, from late spring through early fall in 2003. Outside this period, sampling was limited to unseasonably warm days. Sampling for adults was typically conducted from 1000–1500 h. Population density was estimated by counting the number of adults in three 2.5-m lengths of stream (with a 0.5-m riparian border) during a 5-min period at weekly intervals. Any morphological or behavioral characteristic that was potentially useful in distinguishing males and females in the field was recorded. Observations on adult behavior during flight and mating were noted. Attempts were made to collect adults prior to dusk and overnight on five occasions. An ultraviolet light trap (Model 2851A, BioQuip Products, Inc., Rancho Dominguez, CA) was placed in the riparian zone near the pool concentrated with larvae. Oviposition of O. ocelligera was monitored at weekly intervals from May to August in 2003 and 2004. For each egg mass, we recorded location, microhabitat type, vertical distance above water, number of eggs, size of egg mass, and distance from nearest mass. Ten egg masses were collected at the site for rearing in the laboratory. These were placed in Petri dishes lined with filter paper, moistened with discharge water, and incubated at 12 °C. Embryonic development, hatching, and behavior of first-instar larvae were observed and recorded. Results Physical, chemical, and biotic environment The mean water temperature of the mine discharge as measured at the study pool was 9.3 °C from September to November 2002, and 9.4 °C from March to December 2003 (Table 1). The range of temperature was minimal during those two years: 1.9 °C in 2002 and 1.7 °C in 2003. However, after approximately 90% of the flow was diverted to passive treatment systems in January 2004, the mean water temperature increased to 13.8 °C for the period April to August 2004, and the range of temperature increased to 3.9 °C. Chemical analysis of the mine discharge indicated that the pH was relatively low (2.58–2.99) prior to partial diversion to passive treatment systems in January 2004 (Table 2), and acidity, sulfate, and metals (manganese, iron, 2009 L.A. Redell, W.K. Gall, R.M. Ross, and D.S. Dropkin 291 and aluminum) were relatively high. After diversion of mine drainage, dissolved oxygen and conductivity decreased, temperature increased, and pH slightly increased, ranging from 3.02 to 3.13. Not surprisingly, the fauna of aquatic insects was depauperate in this highly perturbed system, e.g., no species of Plecoptera or Ephemeroptera were present, and O. ocelligera was the only trichopteran collected in the mine drainage before and after diversion. Drift-net samples included a very low diversity of aquatic insects that included Chironomidae (midges), Dytiscidae (predaceous water beetles), Empididae (dance flies), Sialidae (alderflies), and O. ocelligera, as well as terrestrial insects and other arthropods such as Collembola (springtails), Hemiptera (true bugs), Diptera (true flies), Lepidoptera (moths and butterflies), Hymenoptera (bees), Diplopoda (millipedes), and Araneae (spiders). Adult O. ocelligera were collected in drift nets on 3 June 2003 (1 female, 1 male) and 19 October 2003 (1 male). After stream discharge was reduced in early 2004, the presence and temporal distribution of larval instars of O. ocelligera did not differ from patterns observed before diversion in 2002–03. Table 1. Water temperature (°C) measured in the study pool at monthly intervals, 2002–2004. Year Month 2002 2003 2004 March 9.1 April 9.5 11.6 May 9.4 13.9 June 9.4 13.0 July 9.7 15.2 August 9.7 15.5 September 10.4 10.0 October 9.0 9.7 November 8.5 9.2 December 8.3 Mean 9.3 9.4 13.8 SD ± 0.98 ± 0.47 ± 1.61 Table 2. Water quality of Anna S. Mine discharge, 1996–98 (Hedin Environmental 2004) and 2002–04 (USGS). Measurements by the USGS are means of monthly samples taken from July 2002–August 2004, except January and February (see text). Year Physical/chemical variable 1996–98 2002 2003 2004 Water discharge (m3/sec) 0.01 0.01 0.01 ~0.009 pH 2.99 2.58 2.69 3.13 Sulfate (mg/L) 542 Manganese (mg/L) 14 Iron (mg/L) 12 Aluminum (mg/L) 16 Acidity as CaCO3 (mg/L) 271 Dissolved oxygen (mg/L) 9.44 8.89 6.91 Conductivity (μS/cm) 1299 951 758 Temperature (°C) 9.3 9.4 13.8 292 Northeastern Naturalist Vol. 16, No. 2 Eastern Hemlock, which provided about 80% of the canopy at the study site, was the principal riparian vegetation overhanging the pools. Eunotia exigua was numerically abundant in water samples collected from the pool where larvae of O. ocelligera were also sampled; however, it did not constitute a significant component of the biomass compared with Klebsormidium. Eunotia exigua was not epiphytic on Klebsormidium. Larval development A multimodal distribution of head-capsule widths with five peaks, corresponding to five instars, was found for 357 larvae of O. ocelligera collected in kick samples (Fig. 3). The ranges of head-capsule widths for the five instars approximated five normal distributions and are delineated by vertical dashed lines in Figure 3. The number of larvae measured in each instar, the mean, standard deviation, and range of head-capsule widths for each instar, and ratio of means of successive instars, are presented in Table 3. Dyar’s Law predicts that head-capsule widths of insect larvae follow a geometric progression in growth through successive instars (Berg and Merritt 2003). To check whether our delineation of five larval instars for O. ocelligera based on a frequency distribution of head-capsule widths (Fig. 3) conformed to Dyar’s Law, we plotted the log10 of mean head-capsule widths for the five instars (Fig. 4) as suggested by Berg and Merritt (2003). Theoretically, conformity to Dyar’s Law is indicated by a straight line whose slope should be constant for a given species. Our plot in Figure 4 indicates reasonably good conformity to Dyar’s Law, suggesting that we determined the correct number of instars and correct ranges (and resulting means) of head-capsule Figure 3. Frequency distribution of head-capsule widths (mm) of Oligostomis ocelligera larvae collected in acid mine drainage showing ranges for each of the five instars. 2009 L.A. Redell, W.K. Gall, R.M. Ross, and D.S. Dropkin 293 widths for each instar. The slight deviation from a linear relationship between instars I–II, and IV–V, in particular, may be a result of the influence on growth of abiotic factors such as temperature, and biotic factors such as food quantity and quality, in addition to possible errors in measurement of head-capsule widths (Berg and Merritt 2003). Dyar also found that headwidth ratios of successive instars for 28 species of caterpillars were also nearly constant for a given species (mean = 1.5, range = 1.3–1.7; Berg and Merritt 2003). Our calculation of ratios of mean head-capsule widths for successive instars (Table 3) resulted in a mean of 1.50 for the four head-width ratios, and a range of 1.20–1.95, also indicating reasonably good conformity to Dyar’s Law. Oligostomis ocelligera exhibited an asynchronous univoltine life cycle (Fig. 5). Egg masses (Fig. 6A) were observed along the stream channel from 3 June to 12 November 2003. Eggs collected in the field and incubated in the laboratory hatched from 3–14 d at 12 °C. Since first-instar larvae (Fig. 6B) were initially found in August, we infer that eggs hatched in the field Table 3. Mean ± standard deviation and range of head -apsule widths for 357 larvae of Oligostomis ocelligera in instars I–V, and ratio of mean head-capsule widths for successive instars. Instar n Mean ± SD (mm) Range (mm) Ratio of means I 23 0.43 ± 0.05 0.35–0.55 - II 22 0.84 ± 0.08 0.75–1.00 II/I = 1.95 III 94 1.17 ± 0.08 1.05–1.35 III/II = 1.39 IV 173 1.68 ± 0.12 1.40–1.90 IV/III = 1.44 V 45 2.02 ± 0.08 1.95–2.30 V/IV = 1.20 Figure 4. Plot of log10 mean head-capsule widths for the five instars of Oligostomis ocelligera to test conformity to Dyar’s Law. 294 Northeastern Naturalist Vol. 16, No. 2 by mid-summer. Third-instar larvae were numerically dominant by October, although all five larval instars were represented in samples collected during that month (Fig. 5). Instars II–V were still present in December, with samples numerically dominated by fourth and third instars at that time. Only fourth and fifth instars were represented in samples collected from March to July. Behavior First-instar larvae stayed within the liquefying gelatinous matrix of egg masses for several days when reared in the laboratory. After exiting the matrix, larvae immediately began constructing cases. Cases were constructed of leaf pieces arranged in rings. When disturbed, larvae abandoned cases and were observed displacing other larvae to obtain cases. Cannibalism was observed in first-instar larvae reared in the laboratory. Larvae were collected in the mine discharge in pools with an organic substrate of leaves and detritus. When collected with nets from pools, early instar larvae readily abandoned their cases. Larvae were observed crawling on the substrate during mid-summer, and they frequently inhabited leaf packs that were placed in the discharge to provide artificial larval habitat. Occupied pupal cases of O. ocelligera were discovered embedded in soft, decaying submerged logs from 8 April to 10 June. Generally, they were found slightly upstream from pools that contained larvae, in locations where woody debris was concentrated. Logs used by pupae had a mean diameter of 10 cm and a length of 46 cm. Pupal cases occurred individually or in aggregations, with some logs containing as many as 30 cases. During 2003, adults of O. ocelligera (Fig. 6C) were observed along the stream channel from 29 April until 30 October, a remarkably protracted Figure 5. Temporal distribution of larval instars of Oligostomis ocelligera collected at monthly intervals in acid mine drainage. 2009 L.A. Redell, W.K. Gall, R.M. Ross, and D.S. Dropkin 295 Figure 6. A. Oligostomis ocelligera eggs in 5 gelatinous masses (indicated by white arrows). B. Oligostomis ocelligera first-instar larva. C. Adult Oligostomis ocelligera resting on leaf of Betula alleghaniensis (Yellow Birch). 296 Northeastern Naturalist Vol. 16, No. 2 flight period of 185 days or 6 months. Adults were most active during midmorning to early afternoon on warm, sunny days. Few adults were observed on rainy days or at dusk. No adult O. ocelligera were captured in the ultraviolet light trap that was deployed on five nights along the pool where larvae were collected: 1 July, 28 August, and 5, 12, and 20 September. Flights were quick and erratic; adults did not appear to exhibit directional preference for flying upstream or downstream. Most of those captured in flight were males. Adults usually flew directly above the stream channel, but when disturbed, they occasionally flew outside the riparian zone. Initially, we were puzzled by the lack of observations of females. For example, all 16 individuals that we collected between 1145 and 1640 hours on 9 August 2003 were males (included with voucher specimens deposited in the CUIC). Among those 16 males, nine were netted in flight, while seven were netted on (or when flushed from) riparian vegetation. With more intensive searching along the stream channel, females were observed crawling along the mossy stream bank, resting under moss, tree bark or on riparian vegetation, and on a few occasions were discovered underneath submerged logs. Among the three females deposited as vouchers in the CUIC, one was collected while it was walking on leaves along the stream channel on 21 June 2004; two were collected on 19 July 2004, one on a twig in the stream and the other on a leaf of Yellow Birch along the stream channel. Thus, females exhibited more cryptic behavior and/or reduced flight activity than males. During the 2004 field season, pre-copulatory behavior was first recorded on 27 May when adults were observed swarming near waterfalls and crawling on logs and moss along the stream bank. From these waterfall-congregation sites, individuals then engaged in short flights downstream, skimming the water surface several times. On multiple occasions, males were observed resting and then fanning their wings on the water surface. Copulating pairs were observed on four dates in 2004: 3 June (two pairs), 8 June (one pair), 1 July (four pairs), and 8 July (two pairs). No copulating pairs were observed between 8 July and 31 August 2004 when our two-year study was terminated, even though two egg masses were discovered under moss on the latter date. Copulation usually occurred on emergent logs or on the stream bank. Several pairs were observed copulating at mid-day. Members of mating pairs faced in opposite directions, connected by the posterior ends of their abdomens. One pair was observed in copulo on a log for 18 min; that interval was measured from the time of coupling to de-coupling of the two sexes without disturbance by the observer. After copulation, the female crawled away and flew upstream approximately 3 m where she came to rest on the water surface, while the male remained on the log. One post-copulatory female was captured as she crawled on a moss mat on the stream bank. After being placed in a collection jar, she deposited one egg mass before dying. Egg masses were observed along the stream channel from 3 June to 12 November during our two-year study (Fig. 6A). The gelatinous egg masses were oviposited above the water surface in locations with greater than 75% 2009 L.A. Redell, W.K. Gall, R.M. Ross, and D.S. Dropkin 297 canopy cover. Females deposited a single egg mass on the undersides of moss mats, in crevices of logs partly exposed above water in the stream bed, under birch bark, and in the stream bank. In areas with limited habitat for oviposition, egg masses were deposited in clusters. Six egg masses were observed in a 5- x 5-cm section of moss, and ten masses were observed in a 12- x 12-cm crevice of a log. Egg masses (n = 83) were deposited a vertical distance above the water surface ranging from 0.5–24 cm (mean = 6.1 cm). Diet Examination of gut contents of fourth- and fifth-instar larvae revealed a diet of filamentous algae, diatoms (primarily Eunotia), terrestrial mites, detritus, leaf fragments, terrestrial insect fragments, nematodes, and chironomid larvae. Most guts contained both plant and animal material. Filamentous algae, Eunotia, and mites were the principal components found in the guts of larvae. Morphology Gelatinous egg masses were generally pale yellow in color, with white recognizable eggs (Fig. 6A). Sampled egg masses (n = 24) were 3–10 mm in diameter, 9–15 mm in length and contained 31–98 eggs. Microscopic examination revealed that the eggs exhibited a granulated surface with oil droplets. Newly hatched larvae had a highly sclerotized head capsule (Fig. 6B). The mean width of the anterior border of the frontoclypeal apotome was 1.18 mm (n = 21; range = 1.01–1.28 mm) for combined measurements of fifthinstar larvae (n = 11) and larval exuviae recovered from pupal cases (n = 10) (Table 4). Pupae removed from pupal cases were adecticous, i.e., their mandibles lacked a sclerotized apical blade and were reduced to membranous lobes (Wiggins 1998). The length and width of the membranous mandibular lobes were subequal in two pupae collected on 6 May 2004 (deposited as vouchers in the CUIC). The apex of each lobe was slightly rounded and the lateral margin was obtusely angled, with two stout setae inserted dorsally at the lateral angle. In lateral view, the medial portion of the frons exhibited Table 4. Comparison of total length of larvae, and width of the anterior border of the frontoclypeal apotome, of 11 final instar larvae of Oligostomis ocelligera collected 12–18 July 2002 in acidic drainage from the Anna S. Mine, with measurements reported by Wiggins for larvae of O. ocelligera and O. pardalis. Measurement Mean ± SD Range n Total length (mm) O. ocelligera (current study) 19.7 ± 1.7 16.6–21.8 11 O. ocelligera (Wiggins 1960b, 1998) ≈20 Not stated Not stated O. pardalis (Wiggins 1960b, 1998) ≈30 Not stated Not stated Frontoclypeal apotome width (mm) O. ocelligera (current study) 1.19 ± 0.08 1.01–1.28 11 O. ocelligera (larval sclerites removed from 1.18 ± 0.08 1.06–1.26 10 pupal cases; current study) O. ocelligera (Wiggins 1996a, 1998) 1.13 1.13 1 O. pardalis (Wiggins 1996a, 1998) 1.60 1.60 1 298 Northeastern Naturalist Vol. 16, No. 2 a low, convex ridge, which in dorsal and ventral views appeared as a broad but low, conical protuberance. Both pupal cases had a posterior sieve membrane with large pores relative to the narrow silk mesh between them. The anterior ends of both cases were damaged to some degree during collection and/or opening the cases to remove the pupae. However, the pupal case that contained a pharate male of O. ocelligera had an anterior silken mesh still attached to the case by a few silken strands, beyond which were plant fragments loosely interconnected by a tangle of silken strands. Overall, the anterior silken mesh looked rather crude and weak relative to the silken mesh closing the posterior end of the case, i.e., the anterior silken mesh possessed even larger, more irregular pores, and the strands comprising the reticulate mesh were narrower. The presence of an anterior silken mesh could not be determined in the second pupal case, which was less intact at its anterior end. Compared to males, the color pattern of the wings of females was distinctly duller (Fig. 7). Also, the curved yellow band in the hind wing of females was reduced in area relative to the band in the hind wing of males (Fig. 7). Discussion Tolerance of O. ocelligera and other phryganeids to low pH Oligostomis ocelligera inhabited highly acidic mine drainage (pH 2.58– 3.13; from the Anna S. Mine) in which few aquatic organisms can survive. Figure 7. Comparison of wing color pattern, abdomen size, and general appearance of two males and two females of preserved Oligostomis ocelligera: A. Male, 27 June 2003, wingspan (forewings, wingtip to wingtip) = 29 mm. B. Male, 13 July 2004, wingspan = 24 mm. C. Female, 19 July 2004, wingspan = 22 mm. D. Female, 19 July 2004, wingspan = 25 mm. 2009 L.A. Redell, W.K. Gall, R.M. Ross, and D.S. Dropkin 299 Historical water-quality records indicate that fluctuations in water discharge from the mine have little impact on concentrations of sulfate, dissolved manganese and iron, and acidity (Reed 1980). This may indicate that a reservoir of acid water is stored in the mine, either as a pool or as unsaturated ground water. In addition to low pH, the discharge from the mine contained high concentrations of aluminum. Studies have indicated that a combination of pH less than 5.5 and a concentration of aluminum greater than 0.5 mg/L will generally eliminate all fish and many macroinvertebrates (Earle and Callaghan 1998). Few organisms can exist in streams with pH values less than 3.5 and elevated concentrations of iron (Earle and Callaghan 1998). Low pH disrupts the balance of sodium and chloride ions in the hemolymph of aquatic insects (Kimmel 1983). Scattered evidence suggests that among the Trichoptera, phryganeid larvae are the most tolerant of low pH conditions. Roback and Richardson (1969) and Parsons (1968) reported that the phryganeid caddisfly Ptilostomis may be present in streams, impacted by acid mine drainage, with pH values between 3.5 and 4.5. Roback (1974) listed ranges of pH in waters from which 56 taxa of Trichoptera had been collected. The only caddisfly listed by Roback (1974) as occurring in pH less than 5.2 was Ptilostomis sp., which was cited from six records as occurring in waters with a pH as low as 3.3. Ptilostomis was abundant and the only caddisfly present where pH was less than 4.5 and iron greater than 5.0 mg/l (Roback 1974). Fairchild and Wiggins (1989) reported four phryganeids in acidic bog ponds (pH = 4.1) in New Brunswick: Banksiola smithi (Banks), Banksiola crotchi Banks, Banksiola dossuaria (Say), and Agrypnia improba (Hagen). Wiggins and Larson (1989) discovered larval populations of two phryganeids, Beothukus complicates (Banks) and Banksiola dossuaria, in sphagnum bog pools in Newfoundland where the pH ranged from 4.2–4.9. Thus, O. ocelligera joins a group of at least six other species of phryganeid caddisflies whose larvae are tolerant of low pH conditions. By inference, O. ocelligera is among the few caddisflies pre-adapted (in a physiological sense) to tolerate the low pH of the drainage from the Anna S. Mine. It is reasonable to conclude that O. ocelligera has a selective advantage relative to most other caddisflies (not to mention mayflies and stoneflies, which were absent) in this highly perturbed lotic ecosystem. Temperature and the asynchronous life cycle of O. ocelligera What ecological factor(s) might account for the asynchronous univoltine life cycle and the remarkably protracted flight period of O. ocelligera other than the low-pH discharge from the Anna S. Mine? The relatively uniform water temperature in the stream pool (Table 1) where 332 larvae were collected during 2002–2003 may be the primary abiotic factor responsible. Essentially the mine drainage mimics the thermal regime of a typical springbrook, providing very uniform conditions in an area subject to great seasonal changes (Hynes 1970), including establishment of winter-warm conditions. Ward and Stanford (1982) and Ward (1992) point out that some species in 300 Northeastern Naturalist Vol. 16, No. 2 constant temperature springs maintain distinct seasonal cycles, whereas others grow almost continuously throughout the year. The co-occurrence of fourth and fifth larval instars during eight of the ten months that sampling was conducted (Fig. 5) suggests that the population of O. ocelligera in the drainage from the Anna S. Mine represents the scenario of continuous growth throughout the year. Ward and Stanford (1982) cite several studies where emergence of aquatic insects is earlier, and the emergence period is extended, in winter-warm habitats. Thus, the remarkable six-month flight period of O. ocelligera in 2003 in the drainage from the Anna S. Mine provides another example of protracted emergence of an aquatic insect in a winterwarm habitat, albeit a winter-warm habitat that also exhibits low pH. A mechanism that may partly explain the extended emergence of O. ocelligera in the drainage from the Anna S. Mine may be the absence of winter temperatures that are low enough to uniformly stimulate the development of larval diapause (Ward 1992). The diversion of approximately 90% of the flow in the mine drainage to a passive treatment system in January 2004 presented an opportunity to test whether the life cycle of O. ocelligera might shift to a synchronous pattern as a result of the change to a heterothermal regime (Table 1). After diversion in 2004, adults of O. ocelligera were observed along the stream channel between 26 April and 22 July, a shorter flight period (88 days) than in 2003 (29 April–30 October, 185 days) before diversion. However, the opportunity to test the relationship between temperature regime and synchrony of the life cycle of O. ocelligera was not fully exploited, since our two-year study ended on 31 August 2004. The question of what abiotic and biotic factors may have been responsible for the asynchronous life cycle of O. ocelligera in the acid drainage from the Anna S. Mine in 2003 is particularly interesting when one considers that O. ocelligera (as Neuronia stygipes) was reported to emerge only during the last two weeks of May from the headwaters of Argus Brook in the McLean Bogs Natural Area of central New York (Lloyd 1921, Sibley 1926). As far as we know, the studies by Lloyd (1921) and Sibley (1926), limited as they were, provide the only other direct observations on the life history of O. ocelligera. The narrow period of emergence reported by Lloyd (1921) and Sibley (1926) suggests that the population of O. ocelligera at McLean Bogs had a synchronous univoltine life cycle. However, Lloyd (1921) and Sibley (1926) do not provide any data on stream temperature in Argus Brook, and we are not aware of any subsequent studies that do (Robert Wesley, Cornell Plantations Natural Areas Program, Ithaca, NY, pers. comm.). Unlike the drainage from the Anna S. Mine, however, Argus Brook was reported to be alkaline, even though the latter received drainage at times from a bog heath patch (Chamot and Georgia 1926, Young 1926). Biotic factors and the asynchronous life cycle of O. ocelligera Oligostomis ocelligera was the only caddisfly present in the drainage from the Anna S. Mine, whereas stoneflies and mayflies were absent. In 2009 L.A. Redell, W.K. Gall, R.M. Ross, and D.S. Dropkin 301 contrast, Lloyd (1921) and Sibley (1926) recorded at least eight other species of caddisflies inhabiting Argus Brook, viz., Oligostomis pardalis (Walker), Hydatophylax argus (Harris), Phylcentropus lucidus (Hagen), Molanna blenda Sibley, Lepidostoma griseum (Banks), Platycentropus radiatus (Say), Pycnopsyche scabripennis (Rambur), and Frenesia difficilis (Walker). Reduced diversity of aquatic insects in the drainage from the Anna S. Mine may be due to its relative thermal constancy (Ward and Stanford 1982), probably acting in concert with low pH and elevated concentrations of heavy metals. Predicted outcomes of reduced biotic diversity would include reduced competition and reduced predation pressure. Could these biotic factors also have had a causal relationship to the asynchronous life cycle of O. ocelligera in the lowpH, homothermal drainage from the Anna S. Mine in 2003, i.e., could they be at least partly responsible for the protracted flight period, and consequently the extended oviposition and larval recruitment of this caddisfly? Phenology of adults and larval habits of O. ocelligera Adults of O. ocelligera were most active during mid-day from 29 April to 30 October along the stream channel that drains the Anna S. Mine. Diurnal flight of adult O. ocelligera has also been observed in New York (Lloyd 1921), Maine and Nova Scotia (Wiggins 1998), and Tennessee (Etnier and Schuster 1979, Wiggins et al. 2001). Collections of adult O. ocelligera have been reported earlier in the spring in Tennessee than at our study site in north-central Pennsylvania. Etnier and Schuster (1979) collected one male of O. ocelligera in Tennessee on 1 April 1973. Wiggins et al. (2001) collected four males of O. ocelligera with an aerial net in a drainage ditch in Tennessee on 7 April 1998. Larvae of O. ocelligera inhabited pools in the mine discharge that contained considerable leaf litter, consistent with the reports of Lloyd (1921) and Wiggins (1996a). Hamilton et al. (1998) discovered larvae of O. ocelligera in Sarracenia purpurea L. (Northern Pitcher Plant) at Christner Bog in Pennsylvania. They suggested that the larvae entered the pitcher plant leaves in search of food. Karl and Hilsenhoff (1979) collected two larvae of O. ocelligera from detritus in Parfrey’s Glen Creek (pH 7.8), WI, in February. Pupation and the anterior closure of the pupal case of O. ocelligera We found pupae of O. ocelligera embedded in decaying, submerged wood from April to early June in the mine discharge. The pupa of O. ocelligera was described by Lloyd (1921) and Wiggins (1960a, b). Lloyd (1921) reported pupation of O. ocelligera in the stream bed or in dead wood during mid–late April in New York. McGonigle (1987) found pupae of the phryganeid caddisfly Ptilostomis postica (Walker) in burrows and crevices of water-soaked wood, and observed pupal cases stacked end to end in decaying wood on several occasions. As noted by Wiggins (1998), “… pupal cases in all Phryganeidae are effectively concealed — certainly from entomologists, because they are not often found.” Among the seven North American phryganeids known at that time to have degenerate pupal mandibles, Wiggins (1960a) was able to confirm that all 302 Northeastern Naturalist Vol. 16, No. 2 but O. ocelligera closed the anterior end of its pupal case with pieces of plant material fastened loosely together with silken strands. Thus, our discovery of an anterior silken mesh in the pupal case of a pharate male represents the first report for O. ocelligera. However, it does not represent the first report of this condition among the four species of Oligostomis. In his description of the immature stages of O. pardalis (as Neuronia pardalis Walker), Lloyd (1915) stated: “In preparing to pupate the larva attaches its case tightly in some secluded crevice and spins a silken mesh across each end of the case.” Although Wiggins (1998) did not specifically address (or discount) this observation by Lloyd (1915), he states that “… early literature wrongly attributed anterior closure membranes to pupal cases in several genera where evidently they do not exist.” This situation may also apply to the European species, O. reticulata (L.), whose immature stages were described over 100 years ago (Wiggins 1960a). The immature stages of O. soochowica (Ulmer) from China are unknown (Wiggins 1998). Assuming that our finding of an anterior silken mesh in the pupal case of O. ocelligera is confirmed by study of additional specimens, two relevant issues arise: one involves the mechanism for escape of the pharate adult from the pupal case, the other involves evolutionary considerations. We infer that the low convex ridge on the frons of the adecticous pupa of O. ocelligera, although not extended to the same degree as the frontal protuberance on the adecticous pupa of Eubasilissa (Wiggins 1998), may function as effectively in allowing the pharate adult to force its way through both the anterior silken mesh and tangle of plant fragments and silken strands that follow it (Wiggins 1960a, 1998). In the hypothesis of phylogenetic relationships of phryganeid genera proposed by Wiggins (1998), the presence of an anterior silken mesh in the pupal case of O. ocelligera must be interpreted as a reversal to the plesiomorphic condition within the genus Oligostomis (in apomorphic condition, no anterior silken mesh has been constructed, and the pupal case is closed with pieces of plant debris fastened loosely with silk). There is precedent for this evolutionary reversal in behavior: it occurs independently within the genera Oligotricha and Eubasilissa (Wiggins 1998). A reversal to the plesiomorphic state in three phryganeid genera that represent three different phryganeid lineages (Wiggins 1998) suggests that the behavioral repertoire leading to anterior closure of the phryganeid pupal case is evolutionarily plastic or pleiotropic. Oviposition of O. ocelligera and other phryganeids The few observations that have been documented on egg-laying behavior in the Phryganeidae indicate that eggs are embedded in a clear gelatinous matrix and deposited on a submerged substrate (Wiggins 1998). Egg masses of Ptilostomis are sometimes found on damp leaves or logs in basins of temporary pools (Wiggins 1998). McGonigle (1987) suggested that the moisture content of the substrate and wood debris appears to be a major factor in selection of an oviposition site for P. postica. Our observations indicate that O. ocelligera deposits gelatinous egg masses above the water surface in locations with high moisture content. 2009 L.A. Redell, W.K. Gall, R.M. Ross, and D.S. Dropkin 303 Trophic relationships of O. ocelligera and other phryganeids The fauna of low-pH streams is commonly comprised of shredders, collectors, and predators (Earle and Callaghan 1998). Merritt and Cummins (1996) characterized the trophic level of O. ocelligera as both predator and shredder (herbivore and detritivore). Feeding habits of few phryganeid larvae have been studied intensively. Gut analysis of late instars in several genera reveal that larvae feed primarily on aquatic insects, crustaceans, and worms, although algae and vascular plant fragments were also eaten (Wiggins 1996a). We observed cannibalism in early larval instars reared in the laboratory, and stomach analysis of late larval instars collected at the site indicated a diet consistent with that of shredders and predators. Distinguishing final instar larvae of O. ocelligera and O. pardalis Referring to final instar larvae, Wiggins (1996a, 1998) suggested that the width of the anterior border of the frontoclypeal apotome might be a more effective index than total length in distinguishing the two North American species of Oligostomis. Our determination of a mean frontoclypeal apotome width of 1.18 mm based on measurements of 21 final instar larvae or their exuviae recovered from pupal cases of O. ocelligera (Table 4) closely approximates the frontoclypeal width of 1.13 mm measured by Wiggins (1996a, 1998) from the exuvium of a single reared female of O. ocelligera. The range of our measurements for the width of the anterior border of the frontoclypeal apotome of O. ocelligera (1.01–1.28 mm) does not overlap the single measurement of O. pardalis derived by Wiggins (1.60 mm) from the larval exuvium of a single reared female of the latter species. Therefore, our data support the proposal by Wiggins (1996a, 1998) that the width of the anterior border of the frontoclypeal apotome is useful as an index to differentiate final instar larvae of the two North American species of Oligostomis. The mean (19.7 mm) and range (16.6–21.8 mm) for the total length of 11 fi- nal instar larvae of O. ocelligera measured in our study are reasonably close to the approximated total length of 20 mm for final instar larvae of O. ocelligera provided by Wiggins (Table 4). However, compared to frontoclypeal width, total length may not be as effective an index for differentiating final instar larvae of O. ocelligera and O. pardalis due to the inconsistent effects of different fixatives and/or preservatives on musculature, e.g., contraction vs. expansion of larval segments, angle of repose of the head relative to the long axis of the body, etc. In contrast, the frontoclypeal apotome is a sclerotized structure that is not subject to the vagaries of the preservation process. Notwithstanding the above limitation, our data suggest that the approximate total length of final instar larvae provided by Wiggins (1960b, 1998), i.e, ≈20 mm for O. ocelligera and ≈30 mm for O. pardalis, is also useful as an index to differentiate final instar larvae of the two North American species of Oligostomis. Comparison of life histories of O. ocelligera and B. complicatus Among other species of Phryganeidae, Beothukus complicatus exhibits life-history patterns and tolerance to low pH conditions that are similar to those observed for O. ocelligera in our study. Wiggins and Larson 304 Northeastern Naturalist Vol. 16, No. 2 (1989) studied B. complicatus in sphagnum bog pools (pH 4.2–4.9) in Newfoundland, where the population was found to be univoltine, with larvae overwintering as fifth instars and pupating in late May. Adults were observed in June and early July. Examination of stomach contents of B. complicatus larvae revealed mostly filamentous algae and vascular plant fragments, as well as animal material such as insects, crustaceans, mites, and rotifers. Wiggins and Larson (1989) reported that B. complicatus is a noteworthy species because it is one of the few Trichoptera that may be restricted to bog pools of low pH for completion of its life cycle. Voltinism and ecological factors in perturbed lotic ecosystems Knowledge of voltinism and phenology often is necessary for the proper evaluation of field-based population or demographic data (Johnson et al. 1993). Our study indicates that a population of the phryganeid caddisfly, O. ocelligera, has not merely survived, but has flourished in a first-order stream impacted by acid mine drainage. Under conditions of very low pH, high concentrations of heavy metals, and a nearly uniform springbrook-like temperature regime, and liberated from competition and/or predation by Plecoptera, Ephemeroptera, and other species of Trichoptera, O. ocelligera exhibited an asynchronous univoltine life cycle in the drainage from the Anna S. Mine. The remarkably protracted period of flight activity, oviposition, and larval recruitment of this population of acid-tolerant caddisfly provides interesting insights into the relationship between life-history characteristics of an aquatic insect and the abiotic and biotic factors in a highly perturbed lotic ecosystem. Acknowledgments We thank E.L. Lynch, P.L. Chilson, J.R. Redell, B.A. Redell, C.J. Johnson, and C.A. Campbell for their assistance in collecting larval and adult specimens. Sincere thanks to G.B. Wiggins, E.C. Masteller, R.S. Hedin, W.A. Lellis, J.H. Johnson, C.D. Snyder, J.I. Earle, J.C. Cram, and R.E. Hughey for their expert advice on various aspects of our study. We also acknowledge D.F. Charles for identifying the periphyton present in the discharge water; J.A. Macklin for identifying moss samples; L.M. Sumner for measuring larvae, dissecting larval stomachs, and sorting drift net samples; L.P. Nutting for photographing adults of O. ocelligera; and A.N. Redell, who initially discovered O. ocelligera inhabiting the Anna S. Mine discharge during examination of leaf packs for her middle school science fair project. Deposition of voucher specimens of O. ocelligera in the Cornell University Insect Collection was facilitated by J.K. Liebherr and E.R. Hoebeke. Reference to trade names does not imply government endorsement of commercial products. Literature Cited Berg, M.B., and R.W. Merritt. 2003. Growth, individual. Pp. 489–492, In V.H. Resh and R.T. Carde (Eds.). Encyclopedia of Insects. Academic Press, San Diego, CA. 1266 pp. Betten, C. 1934. 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