An example of a possible leech-bryozoan association in freshwater

Abstract Associations of various invertebrate species with bryozoans and sponges are a well-known marine phenomenon but such epizooic communities are far less diverse in freshwater environments. Here an occurrence of numerous leeches Alboglossiphoniacf.papillosa (Braun, 1805), in interstitial spaces between zooids of a colony of the freshwater bryozoan species Plumatellaaff.fungosa (Pallas, 1768) in Eastern Siberia is described. To the best of our knowledge, this record appears to be the first known example of a leech-bryozoan association, although such relationships deserve further research.


Introduction
Marine ecosystems share numerous examples of commensal associations of various invertebrate taxa with sedentary animals such as bryozoans and sponges (Ristedt andSchuhmacher 1985, Wulff 2006). In contrast, the diversity of bryozoan-and spongeassociated epibionts in freshwater is much lower. For example, only a single freshwater shrimp-sponge association has been described so far (Rintelen et al. 2007), while multiple examples of such associations are known from the marine environment (Macdonald et al. 2006). Larvae of a few aquatic insects use freshwater sponges and bryozoans as a food source (Resh 1976, Ricciardi andReiswig 1994), and several mites (Acari: Unionicolidae) occur in parasitic association with freshwater sponges (Edwards and Vidrine 2006). Freshwater bryozoans were found in association with an assemblage of filter feeders, including other bryozoans, sponges, hydroids, and caddisfly larvae (Ricciardi and Reiswig 1994). The interstitial spaces between zooids and around the edges of colonies may be occupied by several invertebrate taxa, i.e. tubicolous rotifers, ostracods, naidid oligochaetes, nematodes, and chironomids (Ricciardi and Reiswig 1994).
Here we describe an occurrence of numerous leeches, Alboglossiphonia cf. papillosa (Braun, 1805) inhabiting interstitial spaces between zooids of a colony of freshwater bryozoan species Plumatella aff. fungosa (Pallas, 1768). We present the results of a molecular and morphological study of both the leech and the bryozoan species and briefly discuss possible explanations of this unusual finding. To the best of our knowledge, it represents the first documented case of a possible leech-bryozoan association.

Materials and methods
A fragment of a willow branch with a bryozoan colony was collected by hand in a shallow coastal site of a floodplain lake in the Lena River basin, Yakutia, Eastern Siberia, Russia ( Figure 1). This sample was preserved in 96% ethanol immediately after collecting. Leech specimens were collected and counted both from the ethanol solution in the vial and from interstitial spaces between the zooids on the basis of careful investigation of the colony under a stereomicroscope (Leica EZ 4D, Leica Microsystems, Germany). The total length of body in each leech specimen was measured using ocular micrometer of the same stereomicroscope.

Results
A bryozoan colony (size of 40×20×25 mm) from Yakutia was heavily invaded by leeches. Twenty-five leeches (Figure 2A, C, D) were found in the interstitial spaces between zooids of this small colony. The living leeches were in spaces between zooids and were not visible externally when first collected ( Figure 2A). The mean length of ethanol-preserved leeches (±s.e.m.) in the sample is 3.9±0.2 mm (min-max = 2.1-6.1 mm; N = 25). The sample includes 10 relatively large specimens of ≥4 mm long (40% of the total sample) ( Figure 2B). The results of molecular and morphological studies reveal that the leeches belong to Alboglossiphonia cf. papillosa (Braun, 1805), while the bryozoan species was identified as Plumatella aff. fungosa (Pallas, 1768). Comments. Current taxonomy of this leech genus is uncertain. Closely related specimens of Alboglossiphonia cf. papillosa with homologous or very similar COI gene sequence (acc. nos. KM095100 and KM095101) (uncorrected p-distance <1%) were collected from the Gusinoye Lake, Yenisei Basin, Eastern Siberia (Kaygorodova et al. 2014). The BLAST search with the 18S rRNA gene sequence returns the nearest members among the Alboglossiphonia taxa from the USA (acc. nos. AF115983 and AY962410) (Apakupakul et al. 1999;Siddall et al. 2005).  (Dash and Vasemägi 2014), but with a low similarity (uncorrected p-distance 3.8%). However, our specimen has 100% accordance with Plumatella fungosa from Austria (acc. no. KC462027) (Hartikainen et al. 2013) by the nuclear 28S rRNA gene sequence.

Discussion
To the best of our knowledge, leeches have not previously been found in association with freshwater bryozoans, although the specific bryozoan colonial structure provides microscopic spaces between zooids supporting suitable microhabitats for diverse epizooic invertebrate communities (Ricciardi andReiswig 1994, Bushnell andRao 1979). Possible causes of leech aggregation within a bryozoan colony recorded by us are unclear. Possible explanations of this unusual finding could include: first, leeches may simply use bryozoans as a shelter. Second, they may also use zooids as a suitable food source, although there are no published observations of this. Third, leeches may be attracted by and feed on other animals inhabiting interstitial spaces between the zooids, e.g., oligochaetes and chironomids, although such animals were virtually absent within this colony. Another possible explanation is that the leeches concentrated within a bryozoan colony because they were recently hatched from the same parent located near this colony. However, the latter hypothesis seems to be unlikely, because Alboglossiphonia cf. papillosa is a small species, the length of which rarely extends 7-8 mm, and adult leeches of smaller size often occur (Lukin 1976). With respect to this, most of the leeches in our sample appear to be adults based on their size, and the shorter lengths of our ethanol-preserved specimens compared to the longer lengths of living leeches measured by Lukin (1976) can be accounted for by the fact that leeches contract in size when preserved.
Although this is the first recorded instance of a leech-bryozoan association, it may represent a largely overlooked phenomenon because of the hidden lifestyle of these small leeches. For example, the Japanese mussel leech Batracobdella kasmiana (Oka, 1910), which has a hidden life style within the mantle cavity of freshwater mussels, has only recently been discovered in the Russian Far East (Bolotov et al. 2017).