Early Life of the Mink Frog (Rana septentrionalis): From Fertilization to Metamorphosis

We collected adult (n = 48 total) and metamorphosing (n = 6) R. septentrionalis from Mercer, Somerset County, Maine on 11 July 2015. Water temperatures recorded on 34 nights in 2000 and 48 nights in 2001 were 19.2±0.2°C on average (range = 18.4–20.1°C; Bevier et al., 2004). A series of adults (n = 35) and all metamorphosing individuals were euthanized for a separate morphological study. We kept 13 adult frogs in 38 L plastic buckets and transported them to Greeley Memorial Lab, New Haven, Connecticut. We then transferred and maintained all frogs in three separate plastic sweater boxes (60 cm x 30 cm x 40 cm) containing half volume of reconstituted deionized water at about 18°C. We housed four or five individuals together in each container (one or two males and three females) that also had pieces of floating foam board insulation (2.5 cm thick) on which frogs could sit (Fig. 1A). We used binder clips to attach strips of fiberglass window screening to each piece of foam. These strips were suspended vertically down the water column to mimic vegetation that R. septentrionalis uses to support egg masses. We fed frogs crickets ad libitum and changed water every 72 hours.

On 13 July 2015, the day after adult frogs were placed in plastic sweater boxes, one egg mass in Group A was produced by natural breeding. This original egg mass predominantly failed (see below) and so we chose to artificially induce breeding in all three groups and raise larvae again. To induce breeding in R. septentrionalis in the laboratory, we used AMPHIPLEX, a mixture of a gonadotropin-releasing hormone agonist (GnRH-A; des-Gly10, D-Ala6, Pro-NHEt9-GnRH) and a dopamine antagonist (metoclopramide hydrochloride; MET) dissolved in saline, following Trudeau et al. (2010, 2013). We injected both adult male and female frogs with AMPHIPLEX intraperitoneally, following published protocols, on 31 July 2015. One egg mass was present the next day in each of two containers.

We maintained embryos from each group in a separate 5 L plexiglass aquaria. Without bubblers, Group A's egg mass dissolved and most embryos perished within a day. With egg masses produced in Groups B and C by artificial breeding, we added bubblers to increase dissolved oxygen, predicting that a lack of oxygen may have killed Group A's naturally produced clutch. Dissolved oxygen measurements from the source pond are relatively high (32–64% at 30 cm below the surface; Bevier, unpubl.) suggesting that embryos of R. septentrionalis require high oxygen levels. The addition of bubblers appeared to have helped Group B and C egg masses survive and maintain structure.

Once hatched and at free-feeding stage (Gosner 25), we fed larvae a mixture of rabbit chow and fish flakes (Lambert, 2015). As larvae grew, we haphazardly split clutches into two or more 5 L aquaria, each with a bubbler, to reduce larval densities. Because we could not predict a priori what larval densities would promote or inhibit development and survival, our attempts to split clutches to reduce densities were not systematic. In mid-April 2016, we moved all remaining larvae to outdoor mesocosms at Greeley Memorial Lab following methods from Skelly (2002), and allowed larvae to develop outdoors.

We sampled larvae from each of the three groups over the year to obtain representative individuals and variation at different developmental stages (Supplemental Table 1; see Data Accessibility). We euthanized larvae in an overdose of MS-222 and fixed them in 10% buffered formalin. Specimens were deposited at Yale Peabody Museum of Natural History and later imaged and analyzed. Snout–vent (SVL) and total length (TL) were measured from digital photos in ImageJ. For hatchlings, images were acquired using a Zeiss Stemi 2000 macroscope with a Q-Imaging Micropublisher 5.0 digital camera. Capture control software (Q-Capture 7) was used to capture multiple images at ascending focus levels (Z-stack) and embed a calibrated scale bar. Images of small larvae were acquired using a Canon 6D DSLR camera mounted on a Leica MZ75 macroscope. Images of large larvae and metamorphs were acquired with a Nikon D90 DSLR and Nikon 60 mm f2.8 macro lens. For DSLR images, Z-stacks were captured with manual focus and merged in Photoshop. Images of scale bars were captured concurrently for calibration. We categorized each tadpole measured to the appropriate Gosner stage (Gosner, 1960; Watkins-Colwell and Leenders, 2004) and then grouped tadpoles of similar stages into one of seven qualitative developmental categories. We present variation in SVL and TL across developmental categories using ggplot2 in R (Wickham, 2016) and use these values, including the proportion of tail length to body length (SVL), to compare to published data from Hedeen (1971) and Altig and McDiarmid (2015).

Several Mink Frogs were observed in amplexus in transit from Maine to Connecticut, and within 24 hours of arriving in the laboratory R. septentrionalis successfully produced one egg mass without intervention (Fig. 1A). This first egg mass was similar in appearance to one collected in the field and photographed (Fig. 1B) and can be compared to egg masses documented in the field (Fig. 1C) as described below. The mass deposited in the lab, however, dissolved and most embryos died within 24 h of oviposition; we hypothesize this was likely due to low dissolved oxygen in laboratory conditions.

AMPHIPLEX injections resulted in three induced egg masses within 24 h, including two masses in Group B and one mass in Group C. We had no negative control treatments for comparison, nor sham controls, so we note that oviposition could have been coincident with AMPHIPLEX injections rather than a result of the injections. Regardless, no oviposition had occurred for about two weeks, between the arrival of R. septentrionalis to the lab and when AMPHIPLEX injections were given, suggesting the injections successfully encouraged both amplexus and ovulation.

All three egg masses produced were oviposited 10–15 cm below the water surface on strips of window screening 3 cm in width and weighted at each end. One egg mass from Group B was fixed in formalin as a morphological specimen and deposited in the Yale Peabody Museum (YPM HERA 019717, Fig. 1D). The other two egg masses were maintained in separate aquaria with bubblers. The addition of bubblers to the aquaria appeared to have enhanced embryo survival and maintained the egg mass structural integrity.

We observed high survival rates under laboratory conditions when bubblers were included in the aquarium set-up. Larvae appeared to grow slowly under initial densities (several hundred animals per 5 L aquarium), so we split each clutch into at least two aquaria to reduce densities to about 200 animals per aquarium. Once they were transferred to mesocosms in March 2016, larvae grew more rapidly. We obtained late stage larvae, including metamorphs, by mid-July 2016 (Supplemental Table 1; see Data Accessibility).

We visually analyzed larvae (Fig. 2, Supplemental Figs. S1–S12; see Data Accessibility), differentiated seven developmental stages across the samples, and plotted these against SVL for each individual (Fig. 3). These different stages are also represented in Figure 4, in which we present TL and plot our lab-reared data in comparison to TL from Hedeen's (1971) field-collected larvae in Minnesota. The proportion of tail length to body length (SVL) is included as a trait for identification of Mink Frogs in Altig and McDiarmid (2015); Figure 5 reflects data collected on the tadpole specimens in our collection to that ratio of 1.6.

Our success in rearing larvae of Mink Frogs in captivity from egg stage through metamorphosis provides important specimens for future study and suggestions for successful husbandry of this species. The egg masses laid in captivity (Fig. 1A, D) are comparable to those seen or photographed under more natural conditions (Fig. 1B, C) and described by Aronson (1943) as a “solid mass or plinth below the surface of the water.” Wright and Wright (1933) include descriptions of egg masses observed attached to vegetation from 20–47 cm (8–18 inches) below the surface of the water. In the field, the egg mass of Mink Frogs consists of 500–4000 black ova with two jelly layers, measuring 75–125 mm in diameter (Altig and McDiarmid, 2015). A mass is attached underwater to vegetation but soon after swells and falls to the bottom of the water body (Hunter et al., 1999). The colder water is likely more oxygenated than the surface water, so it is not surprising that survival of larvae in captivity hinges on highly oxygenated water produced using a bubbler. As Hedeen (1986) suggests, this need for sufficient oxygen to support developing embryos in submerged egg masses, along with low tolerance for desiccation and, possibly, avoidance of predaceous American Bullfrogs where they overlap at the bullfrog's northern range limit, may explain why the Mink Frog exhibits a southern limit to its distribution at 43°N.

Our protocols were far from standardized, but we did our best to provide adequate environments for all life stages. Density, for example, was likely reflected in the range of variation in SVL and developmental stage for larvae from the same clutch sampled at the same time. For example, larvae from Group B removed on 14 and 15 July 2016 exhibited dramatic variation in development (Gosner 25–27 compared to Gosner 42, Supplemental Table 1; see Data Accessibility) despite their kinship. This variation may be an adaptive strategy for variable environmental conditions. For example, if larvae are developing in colder less oxygenated water, or in dense populations, some may overwinter twice as seen in American Bullfrogs (Dodd, 2013). Indeed, larvae of American Bullfrog have extremely variable larval periods and metamorphose in as little as three months for populations in Arizona (Dowe, 1979) or as long as three years in more northern latitudes (Oliver and Bailey, 1939; Bruneau and Magnin, 1980). This results in a range of body sizes at metamorphosis as reported in studies like that of Seale (1980). Berven et al. (1979) report similar variation in Green Frog larval periods, which may extend from 300–670 days in populations at high elevation in Virginia compared to 90–300 days for lowland populations in Virginia and Maryland. We have also observed that larvae may reach only Gosner 25 over a year in a mesocosm (Lambert, pers. obs.). Wilbur and Collins (1973) suggest this variation is consistent for ranid frogs that develop in stable, permanent habitats to ensure larvae reach optimum size for metamorphosis.

Larvae of Mink Frogs are reported to hatch in 5–13 days and grow up to 100 mm TL in 12–15 months (Altig and McDiarmid, 2015). In this study, we collected the first hatched larvae, typically staged 17–20 (Gosner, 1960) at 11 days (Supplemental Table 1; see Data Accessibility). The captive larvae then proceeded through development. Altig and McDiarmid (2015) document that larvae metamorphose at about 42 mm if within the first year of hatching, or 72 mm if they overwinter. We can compare the total lengths and developmental stages of the larvae sampled in this study to those collected from ponds in Minnesota over a year (Hedeen, 1971). Sizes at Gosner stages and the variation seen in both sets of samples are similar (Fig. 4). Mink Frog larvae at different developmental stages are featured in images that show both lateral and overhead views (Fig. 2, Supplemental Figs. S1–S12; see Data Accessibility).

These images augment field guide descriptions, including that from Altig and McDiarmid (2015), which describes mature larvae as having a body with a greenish dorsal surface with brown or black mottling, a medium-sized dorsal fin sometimes with pinkish spots, and a labial tooth row formula (LTRF) of 2/3. There may also be contrasting black marks on the posterior third of the tail at later larval stages (Altig and McDiarmid, 2015). This description also includes the ratio of tail length to body length, which for Mink Frog tadpoles is said to be about 1.6. When we plot this metric for the Mink Frog tadpoles in this study, there is substantial variation both within and between Gosner stages, and only a few individuals even reach this ratio value (Fig. 5). This difference may be the result of comparing our captive-reared tadpoles to those collected and measured from wild populations, and may reflect the various environmental influences on differential growth between body and tail. It is also not clear how this ratio was generated, and to what population(s) of Mink Frogs it is attributed. This trait may be challenging to use to differentiate among sympatric species, but perhaps more investigation would reveal its value. As hind limbs develop in the Mink Frog, it's notable that the third toe is much shorter than the first toe (Altig and McDiarmid, 2015), and webbing extends to the toe tips of the third and fifth toes and to the last joint of the fourth toe. This pattern of webbing is one way to distinguish Mink Frog metamorphs and adults from those of American Bullfrog and Green Frog.

The series of Mink Frog tadpoles we have now documented and analyzed, combined with those for Green Frog and American Bullfrog, contribute a valuable resource to help identify larval stages in the field or in specimen collections. Larval series, including specimens ranging from fertilized egg masses through metamorphs, are relatively scarce, at least in North American museum collections (Altig and McDiarmid, 2015). These series provide rich avenues of research, including opportunities to further investigate potential effects of climate change. Mink Frog occurrence depends largely on water temperature because of the species' sensitivity to thermal conditions at each life stage (Moore, 1952; Hedeen, 1986; Popescu and Gibbs, 2009). Mink Frogs could serve well as an indicator species of climate change, particularly at the southern edge of their range, as conservation biologists are frequently interested in identifying species whose distributional patterns are vulnerable to significant environmental change (Popescu and Gibbs, 2008). There has been extensive research on the influence of climate change on various aspects of the phenology of breeding and activity in the Wood Frog (e.g., Sheridan et al., 2018; Arietta et al., 2020; Larsen et al., 2021). With the reference collection of tadpoles and guidelines for husbandry, the Mink Frog can serve as a different model for studies of climate change on species with more boreal distributions. While Mink Frogs have not generally been considered to be of conservation concern (Casper, 2005; Dodd, 2013), there is growing awareness that a warming climate could impact the species in the southern portions of its range (Popescu and Gibbs, 2009; Patrick et al., 2012). For example, the species is listed as a Species of Greatest Conservation Need in both Maine and New Hampshire's State Wildlife Action Plans. We hope that our descriptions and illustrations of larval Mink Frogs, as well as methodology for their lab rearing, can aid future studies on the species.

Supplemental material is available at https://www.ichthyologyandherpetology.org/h2021133. Unless an alternative copyright or statement noting that a figure is reprinted from a previous source is noted in a figure caption, the published images and illustrations in this article are licensed by the American Society of Ichthyologists and Herpetologists for use if the use includes a citation to the original source (American Society of Ichthyologists and Herpetologists, the DOI of the Ichthyology & Herpetology article, and any individual image credits listed in the figure caption) in accordance with the Creative Commons Attribution CC BY License.

We thank David Skelly for providing laboratory space and use of the live animal room and mesocosms for rearing tadpoles. Thomas Near provided access to the field vehicle used for travel to and from the collection site in Maine. We thank Eric Lazo-Wasem for training in photography techniques and use of photography equipment. Additional material support was provided by the Division of Vertebrate Zoology of Yale Peabody Museum of Natural History. CRB is grateful for funding to support this work from the Natural Sciences Division at Colby College. This work was approved by IACUC (Yale University 2013-10361, 2015-10681, and 2016-10361) and authorized under Maine scientific collecting permit (2015-259).