2018 Southeastern Naturalist Notes Vol. 17, No. 4 N90 A.J. Phillips, B.W. Williams, and A.L. Braswell Observations of Cocoon Deposition, Emergence, and Feeding in Philobdella floridana (Verrill) Anna J. Phillips1, Bronwyn W. Williams2,*, and Alvin L. Braswell2 Abstract - Philobdella floridana, a species of leech in the family Macrobdellidae, occupies a broad, yet seemingly patchy, distribution in the Piedmont and Atlantic Coastal Plain regions of the southeastern US. A recent collection of an adult P. floridana from North Carolina gave us a unique opportunity to observe several poorly understood, and previously mischaracterized, aspects of the ecology of the species, including cocoon deposition, emergence of young from cocoons, and feeding behavior. In addition, we provide new records of P. floridana, extending its known distribution northward in both the Piedmont and Coastal Plain. Our observations provide important insight into the reproduction, diet, and habitat of a widespread yet little understood leech species. The genus Philobdella is part of the family Macrobdellidae, which also includes the North American medicinal leeches (Phillips and Siddall 2005, 2009). Philobdella is not species rich, represented only by Philobdella floridana (Verrill) and Philobdella gracilis Moore. Philobdella floridana is known to occupy a seemingly patchy distribution extending from southern Florida north to southeastern North Carolina, disjunct from its congener P. gracilis, which occurs from southern Mississippi and Louisiana northwards in the Mississippi River Basin to southern Illinois (Moser et al. 2011, Phillips and Siddall 2005, Sawyer and Shelley 1976). As is typical of leeches with no known commercial or medicinal value, basic ecological information is poorly understood for both Philobdella species. The little that is known of the ecology of Philobdella derives in large part from Viosca (1962), who documented his observations of the habitat, behavior, and presumed host affiliation of Philobdella gracile (= P. gracilis) in Louisiana. Viosca reported the species to be most frequently found in mud or under logs at the edge or in shallows of swamps, marshes, bayous, or in canals or ditches. He listed several species of herpetofauna as hosts (i.e., prey, or “victims” sensu Viosca 1962), noting that frog eggs seemed particularly vulnerable to predation by P. gracilis. Viosca suggested that this leech was not known to feed on fishes, humans, or invertebrates, with the rare exceptions of snails an d crayfishes. Our only insight into reproduction in Philobdella species came from a report and illustration of a cocoon discovered under a rock above the water line at the edge of a stream in South Carolina (Sawyer and Shelley 1976). The authors attributed the cocoon to P. gracilis (= P. floridana sensu Phillips and Siddall 2005). Habitat and prey preferences of P. floridana are almost entirely unknown, but are assumed to mirror those reported by Viosca (1962) for P. gracilis. A recent collection of an adult P. floridana from Wake County, NC, provided us with a unique opportunity to document previously unknown—or mischaracterized—aspects of the ecology of the species. Here, we detail cocoon deposition, hatching, and feeding of P. floridana, and update our understanding of the distribution of the species. A.L. Braswell collected a single specimen of P. floridana (Fig. 1) on 30 April 2017 under debris at the edge of a pond, NW of the town of Wendell, Wake County, NC (35.8383°N, 1Department of Invertebrate Zoology, National Museum of Natural History, Smithsonian Institution, 10th Street and Constitution Avenue NW, Washington, DC 20560. 2North Carolina Museum of Natural Sciences, Research Laboratory, 1671 Gold Star Drive, Raleigh, NC 27607. *Corresponding author - bronwyn.williams@naturalsciences.org. Manuscript Editor: Kirsten Work Notes of the Southeastern Naturalist, Issue 17/4, 2018 N91 2018 Southeastern Naturalist Notes Vol. 17, No. 4 A.J. Phillips, B.W. Williams, and A.L. Braswell 78.41671°W). Habitat at the head of the pond was mixed pine/hardwoods. The leech was sampled just below a spring seepage area. We kept the individual alive in a plastic container with leaves, and fed it an earthworm at 3–5-d intervals. We placed a paper towel dampened with spring water in the container to maintain humidity and changed the towel every few days. Approximately 3 weeks following capture, on ~20–21 May 2017, a cocoon measuring 14.2 mm in length, 9.8 mm in width and 7.4 mm in height appeared on the leaf pack (Fig. 1). The cocoon resembled a stiffened ball of foam, superficially similar to that reported for other jawed leech species, e.g., Macrobdella decora (Say), Hirudo medicinalis L., and Haemopis sanguisuga (L.) (Maitland et al. 2000, Moore 1923). We separated the cocoon from the adult leech, placing it and its leaf in a new container; we maintained the humidity as above. On 12 June 2017, we observed a second cocoon, measuring 11.8 mm in length, 8.2 mm in width and 6.2 mm in height, alongside the adult leech, and subsequently transferred it to a separate container. We observed 6 juvenile P. floridana in the container with the first cocoon on 19 June 2017, 3–4 weeks post-deposition. We were unable to observe the container from 13 to19 June; thus, we could not gauge precise length of time between cocoon deposition and hatching. The 6 juveniles were ~1.5 cm in length at rest. We observed a 7th juvenile the following day that was noticeably smaller, ~1 cm in length at rest. On 21 June we counted a total of 10 juvenile P. floridana in the container. On 21 June we introduced a small earthworm, 2–3 mm in diameter, to the container containing the 10 juveniles. One nearby leech responded almost immediately, moving towards the earthworm with rapidity. It attached near the anterior end of the earthworm and remained affixed while the earthworm continued to move around the container. Within minutes, 2 additional juvenile P. floridana had attached to the earthworm (Fig. 2A). After ~20 minutes, the earthworm was noticeably paler in color, and its movements had largely ceased. Several additional leeches latched onto the prey. Approximately an hour after introducing the earthworm to the container, it had been almost entirely consumed (Fig. 2B). Figure 1. Adult Philobdella floridana (NCSM 29800) collected near Wendell, Wake County, NC. Inset shows the first cocoon deposited by this leech in situ. 2018 Southeastern Naturalist Notes Vol. 17, No. 4 N92 A.J. Phillips, B.W. Williams, and A.L. Braswell We first observed hatchlings in the container with the second cocoon on 27 June 2017, ~2 weeks following deposition. Two additional juveniles appeared on 5 July. By 7 July, a total of 5 juvenile P. floridana had emerged from this cocoon. We fixed the 2 cocoons in 80% ethanol ~1 week after emergence of the last juvenile. We also fixed 2 series of juveniles from the first hatch in 80% ethanol: 4 individuals were fixed on 22 June (3 d after hatching), and 3 individuals on 15 July (26 d after hatching). The adult P. floridana was fixed in 80% ethanol on 15 July. We deposited all specimens in the North Carolina Museum of Natural Sciences Non-Molluscan Invertebrate Collection (NCSM 29800–29804). Little is known about the reproductive biology of many leech species, particularly taxa that are not cultured for medicinal or commercial purposes. The only information in the literature regarding reproduction in P. floridana was a report in Sawyer and Shelley (1976) of a cocoon bearing 9 embryos. It is not clear why Sawyer and Shelley (1976) attributed their cocoon to Philobdella, as the illustration (Sawyer and Shelley 1976:80) is wholly inconsistent with those deposited by our captive adult P. floridana, and ironically, given our observations above, resembles the cocoon of an aquatic earthworm. Unfortunately, we have been unable to locate the cocoon reported by Sawyer and Shelley (1976) for examination; although the authors provided a catalog number for this specimen (USNM 51696), we found 2 adult P. floridana, but no cocoon in the associated lot. Cocoons deposited by our captive adult P. floridana were similar in structure and form to those described for several other jawed leech species, including Hirudo medicinalis, Haemopis sanguisuga, Limnatis nilotica (Savigny), and most notably the confamilial Macrobdella decora (Maitland et al. 2000, Moore 1923, Negm-Eldin et al. 2013). Deposition of 2 cocoons in short succession after isolation indicated that sperm can be stored and released in batches for more than 1 fertilization event, although we do not know if this strategy is typical in natural populations, or dependent on environmental cues. Interestingly, half the number of juveniles emerged from the second cocoon as had from the first, and in approximately half the time. As we did not notice any appreciable differences in size of juveniles from the 2 emergence events, we suspect that expedited development offset any benefits that may have been gained from crowding relief inside the cocoon. Emergence from the cocoon did not occur en masse, but rather was staggered over the span of several days. Our collection of P. floridana in Wake County represents only the 4th record of this species from North Carolina, and is the northernmost record of its distribution in the Piedmont Figure 2. (A) Three juvenile leeches attached to the external surface of an earthworm. (B) A cluster of 6 juvenile leeches engorging on what remained of the earthworm after an hour of feeding. N93 2018 Southeastern Naturalist Notes Vol. 17, No. 4 A.J. Phillips, B.W. Williams, and A.L. Braswell physiographic region. The distribution of P. floridana in the Piedmont is poorly understood; nearly all historical records of the species are in the Atlantic Coastal Plain, ranging from just south of the type locality, Lake Okeechobee, FL, north to Lake Phelps, NC (Moser et al. 2011, unpub. record in NCSM Non-Molluscan Invertebrate Collection). A recent collection of P. floridana from Pohick Bay, VA (USNM 1480598), reported for the first time here, extends the known range of the species in the Coastal Plain and in toto. The geographic distribution of P. floridana in its entirety is based on little more than 2 dozen specimens housed in scientific collections and a few unverified published records (Klemm 1982, Moser et al. 2011, Sawyer and Shelley 1976). The paucity of unique locality records of the species likely reflects sampling bias rather than rarity. Recent collections of P. floridana, including those we document here, have been from beneath debris or logs at the edge of a waterbody, consistent with Viosca’s (1962) observations of P. gracilis. These habitats are not typically searched during standard aquatic surveys (e.g., for macroinvertebrates or fishes); leeches encountered during such survey efforts are rarely identified to an informative taxonomic level. Further, P. floridana, similar to its congener P. gracilis, is not known to feed on humans (A.J. Phillips, pers. observ.; Viosca 1962). Consequently, human activity in the water, such as swimming or wading, does not incite predatory behavior in P. floridana as it would for confamilial blood feeders such as Macrobdella spp. As such, we expect that P. floridana is encountered incidentally and infrequently, or via targeted surveys of particular, and undersampled, habitats. In captivity, our adult P. floridana readily fed on live earthworms, an observation that, although seemingly inconsistent with Viosca (1962), is not without precedence. Verrill (1874) noted that the type specimen upon which the original description of Macrobdella floridana (= P. floridana) was based was preserved in the act of consuming a “small lumbricoid worm”. Indeed, earthworms may comprise a substantial component of the diet of P. floridana, given the habitat in which the leeches have been found. Earthworms are typically abundant in damp soils beneath logs or leaf packs in riparian areas. These habitats are also frequently occupied by a variety of herpetofauna, which in turn may provide P. floridana with alternative feeding opportunities. Viosca (1962) listed several species of herpetofauna as hosts for P. gracilis, and it is reasonable to think that similar prey items might be exploited by P. floridana. Although our observations during this study do not explicitly speak to feeding preferences of P. floridana, diet is likely influenced by prey availability. Viosca’s account of prey use of P. gracilis is particularly interesting as it closely resembles those reported for Macrobdella ditetra Moore. Macrobdella species are wellknown for blood feeding from a variety of vertebrate hosts, and have been observed to feed on floating egg masses laid by frogs during spring breeding (Beckerdite and Corkum 1973; Moore 1901, 1953; Smith 1977). Macrobdella ditetra is unusual within the genus because it is not known to feed on humans, a behavior Viosca (1962) reported for P. gracilis. Macrobdella and Philobdella are sister groups within the family Macrobdellidae (Phillips and Siddall 2005, 2009), and it may be that reported similarity in diet results from a combination of phylogenetic similarity and shared habitat, and therefore resources. The distribution of Macrobdella ditetra overlaps that of both Philobdella species (Klemm, 1982). Our observations of feeding behavior of newly emerged juvenile P. floridana are novel, and provide insight into the ecology of this poorly understood life stage. Unlike the adult, which fed by locating and grasping the anterior end of an earthworm, steadily drawing it whole into the mouth, the juveniles were limited by gape size. As such, they attached to the body wall of the earthworm, pierced it, and fed on their prey by sucking internal fluid and tissues through the breached cuticle. This method of feeding is similar to that used by branchiobdellidans, small leech-like annelids that are obligate ectosymbionts primarily 2018 Southeastern Naturalist Notes Vol. 17, No. 4 N94 A.J. Phillips, B.W. Williams, and A.L. Braswell of crayfishes, when presented with prey too large to ingest whole (Gelder and Williams 2015a, b). Multiple juveniles attached to the earthworm soon after it was introduced to the container (Fig. 2A), and as a group, were clearly able to slow the movements of their prey; however, it is likely that this interesting pack behavior was not coordinated or cooperative, but resulted from individual predatory cues. This interpretation is supported by our observations of additional captive leeches when multiple adults have attacked and fed on the same prey item. Acknowledgments. We thank Jan Weems (NCSM) for her support and patience in allowing numerous captive leeches to reside in her kitchen for part of this study, and William Moser (USNM) for providing us with the Pohick Bay record of P. floridana. We are also grateful to Patricia G. Weaver (NCSM) for helpful guidance with manuscript preparation. Literature Cited Beckerdite, F.W., and K.C. Corkum. 1973. Observations on the life history of the leech Macrobdella ditetra (Hirudinea: Hirudinidae). Proceedings of the Louisiana Academy of Sciences 36:61–63. Gelder, S.R., and B.W. Williams. 2015a. Class Clitellata: Branchiobdellida. Pp. 551–563, In J.H. Thorp and D.C. Rogers (Eds.). Thorp and Covich’s Freshwater Invertebrates, 4th Edition, Vol. I: Ecology and General Biology. Academic Press, Amsterdam, Netherlands. 1148 pp. Gelder, S.R., and B.W. Williams. 2015b. Global overview of Branchiobdellida (Annelida: Clitellata). Pp. 628–653, In T. Kawai, Z. Faulkes, and G. Scholtz (Eds.). 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