Chromosome analysis of chironomids ( Diptera : Chironomidae ) – obligatory miners of freshwater sponges ( Porifera )

Karyotypic characteristics of the chironomids Demeijerea rufi pes (Linnaeus, 1761) and Xenochironomus sp., obligatory miners of freshwater sponges, are considered. The karyotype of Xenochironomus sp. is described for the fi rst time. It is assumed that the ordered spatial organization of chromosomes of these species, expressed in terms of the presence of chromocentres of different degrees of stability and an almost complete absence of polymorphism for chromosome rearrangements, represents an adaptation to inhabiting host tissues, i.e. to habitat relative constancy.

The goal of the present work is studying the karyotype of Xenochironomus sp., presumably new to science, morphologically similar to X. xenolabis, and obtaining additional in-formation on cytogenetic characteristics of D. rufi pes.

MATERIAL AND METHODS
Material was collected in water-bodies of the Saratov Region (Table ).Larvae were extracted from the body of the sponge Spongilla lacustris Linnaeus, 1758 when its tissues were dissected in a Petri dish.IV-instar larvae were fi xed in mixture of 96% ethanol and glacial acetic acid (3:1).Identifi cation of species was based on larval morphology (Shilova, 1974;Pankratova, 1983;Pinder, Reiss, 1983).Preparations of polytene chromosomes from salivary gland cells were made by ethyl-orcein technique (Dyomin, Shobanov, 1990).The study of the morphology of larvae and polytene chromosomes was carried out under a microscope with magnifi cations of 15×40 and 15×90.Microphotographs of polytene chro-N.A. Durnova mosomes were obtained using a Practica DC 44 digital photocamera.

RESULTS
Xenochironomus sp. (Fig. 1, a-c; 2, b, d,  f).In the karyotype there are seven chromosome arms that are joined in a chromocentre.Based on the odd number of arms it is supposed that at least one pair of chromosomes is acrocentric.It has not been established which arms comprise other chromosomes, therefore the morphology of chromosomes and their number remains unclear.However, we have revealed small sites similar in structure to the previously studied chromosomes of X. xenolabis (Belyanina, Durnova, 2002), and on the basis of this fi nding chromosome arms of Xenochironomus sp. have been marked.
A description of the karyotypes is given below, based on comparison with the karyotype of X. xenolabis.In arm А there is a small constriction similar to that in section 15 of arm А in X. xenolabis (xenA).In arm B there is a puff (possibly Balbiani ring), similar in morphology to the active region in the section 22 xenB.Additionally, arm B is marked by a thickening restricted on the two sides by a small constriction as in the sections 23-25 xenB.Arm C contains a constriction similar to constriction in section 18 xenC.Arm D contains one nu-cleolus (N), situated close to the chromocentre; the dark discs on the distal part of this arm are similar to discs in sections 27-28 xenD.In arm Е no common markers for the two species have been revealed; in Xenochironomus sp. this arm is marked by three dark disks on the distal end.In arm F in Xenochironomus sp. a large puff and three distinct discs near it as in the sections 5-7 xenF are situated.Arm G in the karyotypes of both the species is the shortest and thinnest.
When cytological slides were prepared using the method of squashing the chromocentre was retained in the majority of cells of Xenochironomus sp., but in approximately 20% of cases arm D containing a nucleolus separated from it (Fig. 1, a).Sometimes different combinations of two arms (Fig. 1, b, c) occurred with approximately similar frequency, and the most preferable associations of arms have not been revealed.
In few larvae a small heterozygous inversion was revealed in arm A close to chromocentre.In arms B, C, and G there are areas with partially destroyed conjugation of homologues, which is probably a consequence of heterozygous microrearrangements.
In spite of the great similarity of larvae of Xenochironomus sp. and X. xenolabis, some morphological differences between them have   been revealed.In Xenochironomus sp.close to the large median denticle of mentum usually there are small denticles (Fig. 2, b), exterior ones according to some authors (Pinder, Reiss, 1983;Makarchenko, 2006) but fi rst lateral ones according to other authors (Shilova, 1974;Pankratova, 1983).In X. xenolabis these denticles in the majority of cases are completely reduced (Fig. 2, a).In X. xenolabis the seta beneath the denticles of the mandible is slightly extended in the middle part (Fig. 2, c), whereas in Xenochironomus sp. it is thinner, smoothly narrowing towards the apex (Fig. 2, d).In Xenochironomus sp. the sensilla of the antenna nearly reaches the end of the third segment (Fig. 2, f), whereas in X. xenolabis it terminates in the lower half of the fourth segment (Fig. 2, e).
Demeijerea rufi pes (Fig. 3, a, b).In the karyotype there are 6 chromosome arms joined in a chromocentre.It has not been established what particular arms comprise individual chromosomes, therefore the morphology and chromosome number have not been clarifi ed.The polytene chromosome disc pattern conforms to the earlier published data (Belyanina, 1993).
In the present study measurement has been carried out for the fi rst time; and designation of chromosome arms has been made beginning with the longest arm: rufA (IR)> rufB (IIIR)>rufC (IIR)>rufD (IL)≥rufE (IIL)>rufF (IIIL).When cytological slides were prepared with the squashing method the chromocentre was retained in the majority of cells (Fig. 3, а), but in some cases (approximately 10-15% of cells) its structure was disturbed and arms were separated, lying either separately, or in the form of associations, mostly in combinations B+D (Fig. 3, b), A+F, C+E (not shown).Chromosome rearrangements have not been recorded.

DISCUSSION
The shared characteristic of the karyotypes of Xenochironomus sp. and D. rufi pes is the ordered arrangement of chromosomes in salivary gland cells caused by the presence of a single chromocentre of differing degrees of stability (more stable in Xenochironomus sp. as compared to D. rufi pes).This phenomenon was earlier described in X. xenolabis (Belyanina, Durnova, 2002).For D. rufi pes preferable associations of chromosome arms (AF, BD, CE) have been established that were observed when the single chromocentre was destroyed.It is assumed that D. rufi pes has 2n=6 (Belyanina, 1993), but the study of the metaphase plate is required to determine the exact chromosome number of this species.In Xenochironomus sp. and X. xenolabis mechani-   Belyanina (1993).The other designations as in Fig. 1.Bar=10 μm.N.A. Durnova cal destruction of the chromocentre in rare cases led to accidental combinations of two arms.Analysis of metaphase chromosomes of X. xenolabis has shown that this species has 2n=8 (Belyanina, Durnova, 2002).It can be assumed that closely related Xenochironomus sp. also has 2n=8, but this supposition needs corroboration.The polytene chromosomes of Xenochironomus sp. as compared to X. xenolabis are characterized by a specifi c pattern of discs and localization of active regions.
The functional role of spatial arrangement of chromosomes in nucleus has not been established (Zhimulev, 1992).It is known, however, that the presence of a chromocentre leads to ordered spatial arrangement of chromosomes in the interphase nucleus and probably to restriction of variation of their structure (Stegniy, 1993).Joining of centromeres into a single chromocentre is regarded as a primitive state and disappearance of the chromocentre as an evolutionary tendency characteristic of the family Сhironomidae and other Diptera (Stegniy, 1993;Shobanov, 2002).
The species considered in the present article are narrowly specialized and they are bound by close biotic relations with freshwater sponges.It can be suggested that the presence of a chromocentre testifi es to a relative evolutionary primitiveness of these species.The absence (or low frequency) of chromosomal rearrangements is apparently connected with the relatively constant habitat conditions of larvae inhabiting sponge tissues.
At the time of the present only one species of the genus Xenochironomus -X.xenolabis, which has been reported also for Russia, is known from the Holarctic (Zorina, 2006).This study performed shows that Xenochironomus sp. has notable differences from X. хenolabis in both karyotype and larval morphology.This allows the assumption that Xenochironomus sp. is a new species for Russia, and possibly also to science.However, this could be stated more convincingly after all the stages of metamorphosis, and primarily adult male morphology are studied.
Fig. 1, a-c.Karyotype of Xenochironomus sp. a -common chromocentre is not destroyed.b, c -chromocentre is destroyed; b -combination of arms AB and arm D are separated from chromocentre; c -combination of arms BE and arms A and G are separated from chromocentre.A, B, C, D, E, F, G -chromosome arms.Marker features of chromosome arms are designated by square brackets.N -nucleolus, p -puff, BR -Balbiani ring.Bar=10 μm.

Table .
Collecting data.