Freshwater sponge-dwelling Chironomidae ( Insecta , Diptera ) in northeastern Brazil

The Neotropical region possesses a rich freshwater sponge fauna; however, knowledge about its taxonomy and the community inside the sponges remains fragmentary. Chironomidae (Insecta, Diptera) larvae associated with freshwater sponges have been largely reported in literature and some genera are recognized as containing species exclusively dependent on these organisms. In this study, we analyze the occurrence of chironomid larvae associated with freshwater sponges (Spongillidae) in northeastern Brazil. Samples were collected in two aquatic systems in Pernambuco, Brazil, in February 2011 and October 2012. In the Rio Sirinhaém, Corvoheteromeyenia australis (Bonetto & Ezcurra de Drago) was the only sponge collected, and Polypedilum Kieffer, Goeldichironomus Fittkau, Xenochironomus Kieffer and Cricotopus Van der Wulp were the chironomids found as its associated fauna. On the other hand, in the ishpond, Heteromeyenia cristalina Batista, VolkmerRibeiro & Melão, Radiospongilla inesi Nicacio & Pinheiro and Tubella variabilis Boneto & Ezcurra de Drago were the collected sponges, and Polypedilum, Chironomus Meigen, Dicrotendipes Kieffer and Goeldichironomus were the larvae associated with these species. Although only species of Xenochironomus are reported to have close interactions with freshwater sponges, herein we document other Chironomidae genera associated with these organisms. Nevertheless, further investigations are needed to better understand how chironomid larvae use sponges as shelter and/or to avoid predation in a facultative way. The objective of the present study was to analyze the occurrence of Chironomidae inhabitants of freshwater sponges in aquatic systems in the state of Pernambuco, in order to contribute to the knowledge of sponge-dwelling chironomids in northeastern Brazil.


Study area
The Rio Sirinhaém originates in the municipality of Camocim de São Félix, Pernambuco, Brazil, and extends about 158 km in a northwest-southeast direction.It also crosses the municipalities of Gameleira and Cortes, where part of this study was conducted (08°30'S, 35º31'W).The second sampling site was in a ishpond located on the campus of the Federal Rural University of Pernambuco (8°01'S, 34°56'W).This system presents different types of sediments (sand and clay) at the bottom and is formed by water lowing from Plata Creek, located in Parque Dois Irmãos, in northwestern Recife, which had its course changed to supply the ishpond to farm tilapia for scientiic research.

Collection and identiication
Collections were made in February 2011 in Rio Sirinhaém and in October 2012 in the ishpond.In each of these environments, we searched for sponges on several potential substrates, such as decaying wood, rocks, and macrophytes.All samples were ield-preserved using 70% ethanol.In the laboratory, the sponges were dissected and slide-mounted following the procedures described in Hajdu et al. (2011).Identiications were made by the team of the Laboratory of Porifera, Federal University of Pernambuco, based on Manconi and Pronzato (2002) and Nicacio and Pinheiro (2015), mainly using sponge spicules.Larval chironomids associated with different sponges were sorted using a stereomicroscope and slide-mounted following the procedure outlined by Pinder (1983).The material was morphologically identiied to the lowest taxonomic level possible based on the keys in Wiederholm (1983), Epler (2001), Andersen et al. (2013), Trivinho-Strixino (2014) and Silva et al. (2018), and occasionally original descriptions.Gut content examination was made according to Mcshaffrey and Olive (1985), based on previously prepared slides of larvae.No attempt was made during collection to prevent regurgitation of food in the gut; although, all analyzed larvae had some gut content.Guts were left in the bodies and examined microscopically by transparency through the cuticle (for more details see SILVA et al., 2008a;2008b).Voucher specimens are deposited in the Laboratory of Assessment, Recovery, and Restoration of Aquatic Ecosystems, at the Federal University of Pernambuco.

Results
In total, 266 representatives of subfamilies Chironominae and Orthocladiinae were recorded inhabiting four species of freshwater sponges (Table 1).Six genera were identiied.Polypedilum Kieffer and Goeldichironomus Fittkau were the most abundant genera, predominating in all samples.Xenochironomus was only sampled in the Rio Sirinhaém, while Chironomus Meigen and Dicrotendipes Kieffer occurred entirely in the ishpond.Only one genus was recorded for Orthocladiinae, Cricotopus Van der Wulp.In the Rio Sirinhaém the chironomid fauna were exclusively associated with Corvoheteromeyenia australis (Bonetto; Ezcurra de Drago).Other species of freshwater sponges were not found in this environment.On the other hand, in the ishpond, Chironomidae larvae were collected in association with Heteromeyenia cristalina Batista, Volkmer-Ribeiro & Melão, Radiospongilla inesi Nicacio & Pinheiro and Tubella variabilis Bonetto & Ezcurra de Drago.This aquatic ecosystem presented not only the highest diversity of freshwater sponges, but also the highest abundance of chironomids (Table 1).In general, the gut content of the analyzed larvae was predominantly diatoms and fine organic matter.Spicules of H. crystalina were found in larvae of Goeldichironomus, while spicules of C. australis were observed in larval Cricotopus.

Chironomus Meigen, 1800
Chironomus is a cosmopolitan genus with several hundred species.Numerous species of this group are very abundant in heavily polluted standing or running waters, whereas some species usually colonize small water bodies, such as ish breeding tanks, where they complete their life cycle in a few days (CORREIA et al., 2013).Spies and Reiss (1996) catalogued 19 species from the Neotropical region.However, this number clearly represents outdated knowledge and signiicantly underestimates the real diversity of Chironomus in the Neotropics, considering that several new species have been described more recently (CORREIA; TRIVINHO-STRIXINO 2005;2007;CORREIA et al., 2005;2006;2013).In our study, larvae of Chironomus (see Figure 1a) were found associated with the following freshwater sponges: Corvoheteromeyenia australis (Bonetto; Ezcurra de Drago), Heteromeyenia cristalina Batista, Volkmer-Ribeiro & Melão, Radiospongilla inesi Nicacio & Pinheiro and Tubella variabilis Boneto & Ezcurra de Drago.None of the larvae analyzed in the present study had any spicules in their digestive tracts, which were illed with debris, sand and diatoms.

Cricotopus Van der Wulp, 1874
This genus is one of the largest in subfamily Orthocladiinae, comprising ive subgenera, and has a worldwide distribution (CRANSTON et al., 1989;ASHE;O'CONNOR, 2009).Larval Cricotopus are known to inhabit a wide range of water bodies, from pristine streams and brooks to eutrophic ponds and brackish estuaries (HIRVENOJA, 1973;BOESEL, 1983).Certain species may become so abundant in eutrophic waters that adult swarms become a nuisance (SPIES, 2000;HIRABAYASHI et al., 2004).The group is often recorded in association with aquatic macrophytes, algae and sometimes cyanobacteria.Some larvae mine in macrophytes, while many graze on the surfaces (CRANSTON et al., 1983), and according to Roback (1968) they are capable of living in and on sponges as well as other habitats.In our study, larvae of Cricotopus (Figures 1b; 1c) were associated with Corvoheteromeyenia australis.The digestive tract was illed with debris and spicules of the aforementioned freshwater sponge (Figure 1d).

Dicrotendipes Kieffer, 1913
Dicrotendipes is a large group with a worldwide distribution.It is commonly found in littoral sediments of brackish and fresh water, in lotic and lentic conditions, in pristine or degraded habitats (EPLER, 2001), and in many cases it is the most abundant organism.Although larvae occur in sediments, they are most often encountered on vegetation (EPLER, 2001).The group is considered pollution-sensitive to most forms of toxic pollution but thrives in areas containing high levels of nutrients or organic wastes (SIMPSON;BODE, 1980).Its most common associates in such situations are oligochaetes.In our study, larval Dicrotendipes were found in association with Heteromeyenia cristalina and Radiospongilla inesi (Figure 1e).None of the larvae analyzed in the present study had any spicules in their digestive tracts.This might be due to the fact that the larvae here examined may not have achieved their full body growth, since it seems that the presence of spicules in the gut of sponge-dwelling chironomid mainly occurs in inal instars (ROBACK, 1968).

Goeldichironomus Fittkau, 1965
Goeldichironomus is a Pan-American genus, which commonly occurs in eutrophic standing and slowmoving water, where it is found in or on sediments and aquatic plants (EPLER, 2001).Immatures of this genus favor loating and drifting vegetation of small standing water bodies.Several species are miners that form small tunnels in the petioles of macrophytes and decaying wood (REISS, 1974;TRIVINHO-STRIXINO, 2014).They also live in burrows in plant material made by other organisms (EPLER, 2001).The larvae analyzed in our study were identified as Goeldichironomus neopictus Trivinho-Strixino and Strixino (Figure 1f).These were the most common larvae and were encountered in association with Corvoheteromeyenia australis, Heteromeyenia cristalina and Radiospongilla inesi.Larval Goeldichironomus were found throughout the body of the sponge.The digestive tract contained many sponge spicules of H. cristalina (Figures 1g;1h), indicating that the larva was probably feeding on this sponge and its presence may not have been merely accidental.

Polypedilum Kieffer, 1912
This genus is one of the largest in the Chironominae and has eight subgenera, a worldwide distribution, and about 440 described species (SAETHER et al., 2010).Larvae of Polypedilum occur in nearly all types of still and lowing waters, ranging from pristine to heavily degraded, except for in the arctic and high mountains.Sediments are the preferred substratum.Some species are also found on hard substrata, as well as mining aquatic plants and in bromeliad tanks (CRANSTON;EPLER, 2013).Larval Polypedilum are ilter-feeders, and their occurrence seems to be governed more by current speed and the amount of suspended food particles than by water quality (SIMPSON;BODE, 1980).The immatures analyzed in our study were identiied as Polypedilum (Asheum) sp.Sublette (Figures 1i;1j), which is commonly found in lakes, reservoirs and rivers, mainly in association with aquatic macrophytes (TRIVINHO-STRIXINO, 2014).The taxon was previously recorded for southeastern Brazil by Silva and Farrell (2017).Herein, the species was found interacting with Corvoheteromeyenia australis, Heteromeyenia cristalina and Radiospongilla inesi.None of the larvae examined had any spicules in their digestive tracts, which were basically illed with debris.

Xenochironomus Kieffer, 1912
This is a small genus with species distributed in the Afrotropical, Nearctic and Neotropical regions.Larvae of Xenochironomus are obligate miners in freshwater sponges in both standing and lowing waters (CRANSTON; EPLER, 2013).The immatures of Xenochironomus can be found in freshwater sponges with the exception of Xenochironomus canterburyensis (FREEMAN, 1959), which has been reported in association with Mollusca (FORSYTH;MCCALLUM, 1978).This is the irst record of Xenochironomus from the Northeast Region of Brazil.The larvae have very large salivary glands and line the tubes they create with silk as they move along (ROBACK, 1968).The immatures analyzed here were identified as Xenochironomus (Figures 1k; 1l) and were found associated only with Corvoheteromeyenia australis.Some were in gently curved tubes, both ends of which opened on the surface of the sponge, and others were in the internal cavities of the sponge.None of the larvae found in the present study had any spicules in their digestive tracts.

Discussion
Spongillidae has an interspeciic relationship with the family Chironomidae.In our study, we record for the irst time the larvae of Polypedilum, Chironomus, Cricotopus, Dicrotendipes and Goeldichironomus associated with freshwater sponges.To most insects, such as baetid and heptageniid mayfly nymphs, hydropsychid caddisfly larvae, as well as larval chironomids, the sponges seem to be merely another substrate (ROBACK, 1968), since there was no evidence indicating an obligate association of chironomids with the sponges, despite the fact that larvae were found living within the sponges and/or feeding on them.However, the association between chironomid larvae and freshwater sponges might beneit the chironomids by improving feeding opportunity, providing protection from disturbances and decreasing predation risks.This appears to be the scenario in the ishpond, where the association of larval chironomids with sponges may be related to a defensive strategy and/or predation avoidance (mainly from tilapia), since these taxa were not found free-living or on the bottom and walls of this environment.Moreover, the higher diversity of sponges in the ishpond suggests that this environment provides more adequate conditions for the development of these organisms, which may be related to the lentic character of the system compared to the Rio Sirinhaém with lotic characteristics and lower diversity.
Despite the presence of spicules in the gut content of some examined larvae, this association might be considered facultative, since except for Xenochironomus, all the other genera are widely recorded in different environments.Furthermore, the amount of spicules observed does not seem to be enough to assure this association as an obligate relationship, since spicules were found associated with other types of food, such as detritus and diatoms, which are commonly recorded in the Chironomidae diet.Roque and Trivinho-Strixino (2005) described Xenochironomus ceciliae as a freshwater spongedwelling Chironomidae in the upper Paraná River.We recorded larvae of Xenochironomus in association with C. australis.Moreover, the water quality of the Rio Sirinhaém, which is rich in ine particles of organic matter, seems to be suitable for the development of the sponge and the observed interspeciic interaction with chironomids.Larvae of Xenochironomus have a brush of numerous long labral setae, which indicates a ilter-feeding habit, likely related to the particles originating from the aquifer system of sponges (ROQUE; TRIVINHO-STRIXINO, 2005).
When compar ing our results with Roback (1968) and Fusari et al. (2008), who recorded several larvae of Chironomidae associated with different species of freshwater sponges, it is possible that some species of the following genera might still be found interacting with sponges: Oukuriella Epler, Orthocladius Va n der Wulp and Stenochironomus Kieffer.Even though our inventory documents 266 representatives of two subfamilies living in four species of freshwater sponges, we consider this a small freshwater spongedwelling chironomid community for northeastern Brazil.We believe that collections in different periods and broadening the variety of sampling habitats and geographic area may reveal a greater diversity than currently recorded.Despite the increased activity in studying the Neotropical chironomid fauna in recent years, data on the interaction between chironomids and freshwater sponges remain fragmentary.Thus, additional inventories are needed to improve our understanding of the insects that might be associated and to add to the information about those already known to be associated.Studies on interspeciic interactions between chironomids and sponges remain incipient and, due to a lack of information, the relevance of obligatory or optional associations for Spongillidae and Chironomidae larvae is still unclariied.This study reports for the irst time the larvae of Polypedilum, Chironomus, Dicrotendipes and Goeldichironomus living inside freshwater sponges.Despite that the association between these organisms might beneit the chironomids by improving feeding opportunity and providing protection, no evidence indicates an obligate relationship between these taxa and the different species of freshwater sponges recorded.
Freshwater sponge-dwelling Chironomidae in northeastern Brazil we analyze the occurrence of chironomid inhabitants of freshwater sponges in aquatic systems in the state of Pernambuco, Brazil.
Freshwater sponge-dwelling Chironomidae in northeastern Brazil