Chironomus riparius (Diptera) genome sequencing reveals the impact of minisatellite transposable elements on population divergence

Active transposable elements (TEs) may result in divergent genomic insertion and abundance patterns among conspecific populations. Upon secondary contact, such divergent genetic backgrounds can theoretically give rise to classical Dobzhansky–Muller incompatibilities (DMI), thus contributing to the evolution of endogenous genetic barriers and eventually causing population divergence. We investigated differential TE abundance among conspecific populations of the nonbiting midge Chironomus riparius and evaluated their potential role in causing endogenous genetic incompatibilities between these populations. We focussed on a Chironomus‐specific TE, the minisatellite‐like Cla‐element, whose activity is associated with speciation in the genus. Using a newly generated and annotated draft genome for a genomic study with five natural C. riparius populations, we found highly population‐specific TE insertion patterns with many private insertions. A significant correlation of the pairwise F_ST estimated from genomewide single‐nucleotide polymorphisms (SNPs) and the F_ST estimated from TEs is consistent with drift as the major force driving TE population differentiation. However, the significantly higher Cla‐element F_ST level due to a high proportion of differentially fixed Cla‐element insertions also indicates selection against segregating (i.e. heterozygous) insertions. With reciprocal crossing experiments and fluorescent in situ hybridization of Cla‐elements to polytene chromosomes, we documented phenotypic effects on female fertility and chromosomal mispairings. We propose that the inferred negative selection on heterozygous Cla‐element insertions may cause endogenous genetic barriers and therefore acts as DMI among C. riparius populations. The intrinsic genomic turnover exerted by TEs may thus have a direct impact on population divergence that is operationally different from drift and local adaptation.     

Site specific differentiation in metal tolerance in the midge Chironomus riparius (Diptera,Chironomidae)

Metal tolerance in Chironomus riparius (Diptera) populations from contaminated streams was studied by comparing the effects of cadmium, zinc and iron on first generation laboratory reared midges. First instar larvae were exposed for four days, after which surviving larvae were counted and their length measured. Larvae from two highly polluted sites, kept under control conditions, grew substantially slower than those from other populations. All populations showed the same growth responses to increased zinc concentrations, but differences were found in the responses to both cadmium and iron. Since population differentiation was demonstrated in first generation laboratory animals, it is suggested that the differences between populations of C. riparius have a genetic basis.     

A structural study of the anal papillae of the midge Chironomus riparius Meigen (Diptera: Chironomidae)

Larvae of the midge, Chironomus riparius Mg., have four anal papillae arranged as two pairs, one dorsal to and the other ventral to the anus. Structural study with light and electron microscopes has revealed that their integument consists of a thin cuticle overlaying a thick, syncytial epidermis which is specialised to facilitate ion transport. There is a distinct neck or collar region at the junction of each papilla with the rest of the body wall. Although in many respects these structures resemble those in mosquitoes, there are both morphological and physiological features which suggest that differences exist in the means by which ion exchange with the environment is controlled and effected in the two families.     

An ultrastructural study of the larval integument of the midge,Chironomus riparius meigen (Diptera: Chironomidae)

Summary The larval integument of the midge, Chironomus riparius Mg., is unusually thin although it conforms with the normal insect pattern. The cuticle of the post-cephalic segments is about 3 m thick and overlies an epidermis which has an irregular basal plasma membrane resulting in spaces occurring between it and the basement membrane. The ventral tubuli have a similar epidermis but the cuticle is somewhat thinner. The anal papillae have the thinnest cuticular covering with a uniquely folded epicuticle of variable thickness, and their epidermis has the characteristics of a transporting epithelium. No evidence of pore canals could be found in the cuticle of any part except the head capsule which has a remarkably smooth epicuticle and a distinct layer which may represent the exocuticle. There are no spaces between the basement membrane and basal plasma membrane of the epidermis in the head. Ultrastructural evidence would suggest that gaseous exchange can occur across most of the post-cephalic integument.The author is indebted to Mrs. L. Rolph and Mr. R.L. Jones for their technical assistance     

The ultrastructure of the non-neurosecretory components in the brain of the midge,Chironomus riparius Mg. (Diptera: Nematocera)

The ultrastruct of the neural sheath, glial cells and neurons in the brain of the neoimaginal male Chironomus riparius is described. The neural sheath comprises a neural lamella and underlying perineurium. The neural lamella consists of an amorphous matrix in which fine fibrils occur. The perineurium is composed of two cell types forming a continuous layer around the brain. The subjacent cortical layer, composed of the cell bodies of neurons and glial cells, varies considerably in thickness and surrounds the centrally located neuropiles. Three types of glial cells are distinguished on the basis of their positions and appearances. Five types of neurons are described which differ in size and relative frequency of organelles. Four types of axons, including those of neurosecretory cells, are distinguished by their size and content.     

Swimming kinematics and wake elements in a worm-like insect: the larva of the midge Chironomus plumosus (Diptera)

The kinematics and hydrodynamics of swimming chironomid larvae were investigated with the aid of videography and dye streamers used to visualize near-body flow. Chironomids employ a characteristic 'figure-of-eight' swimming technique based on high-amplitude side-to-side bending of the body. These scissor-like movements produce relatively slow (two body lengths (BL) s⁻¹) forward motion but also serve to support the weight of the insect against its own negative buoyancy. The main wake element identified by the present technique consisted of a discrete ring vortex with an external diameter of c. 0.3 BL which was shed to the rear of the body towards the end of each half-stroke. During level swimming, the jet of the vortex was directed 10° below the horizontal plane indicating that it was mainly providing thrust. An additional, but poorly defined, flow was associated with the rapid downwards motion of the head at the start of each half-stroke and it is proposed that this contributes to the vertical force needed to support the weight of the body during swimming.     

Development and localization of microsatellite markers for the sibling species Chironomus riparius and Chironomus piger (Diptera: Chironomidae)

Five variable microsatellite loci are reported for the nonbiting midge species Chironomus riparius and Chironomus piger. All loci show considerable intraspecific variation and species‐specific alleles, which allow to discriminate among the two closely related species and their interspecific hybrids, and to estimate genetic diversity within and between populations. Additionally, the loci were localized on C. riparius polytene chromosomes to verify their single copy status and investigate possible chromosomal linkage. The described markers are used in different studies with regard to population and ecological genetics and evolutionary ecotoxicology of Chironomus.     

Proteomic evaluation of cadmium toxicity on the midge Chironomus riparius Meigen larvae

Heavy-metal pollution of aquatic ecosystems is a widespread phenomenon after industrial consumption. Whether aquatic organisms are adapted to the heavy-metal pollutants or not, such environmental stress causes changes in physiological responses. In this study, the aquatic midge, Chironomus riparius Meigen, was used to find changes of expression of proteins in relation to cadmium exposure. Dose-response relationships between cadmium concentrations and mortality of 3rd instar midge larvae were observed and the protein levels were compared using PD-Quest after 2-DE. Comparing the intensity of protein spots, 21 proteins decreased and 18 proteins increased in response to cadmium treatment. With increased proteins, three enzymes such as S-adenosylmethionine decarboxylase, O-methyltransferase, and aspartokinase were involved in the glutathione biosynthesis and a key enzyme regulating fatty acid biosynthesis, oleyl-acyl carrier protein thioesterase was also identified. According to the functional classification of decreased levels of proteins, they were involved in energy production, protein fate, nucleotide biosynthesis, cell division, transport and binding, signal transduction, and fatty acid and phospholipid metabolism in the cell. In addition, phenol hydroxylase, thioesterase, zinc metalloprotease, and aspartate kinase were newly expressed after cadmium exposure at the concentration of the LC(10 )value. Therefore, these proteins seem to be potential biomarkers for cadmium exposure in the aquatic ecosystems.     

Inherited and somatic cytogenetic variability in Palearctic populations of Chironomus riparius Meigen 1804 (Diptera, Chironomidae).

Inter- and intracytogenetic variability was analyzed in 13 natural Palearctic populations of Chironomus riparius Meigen 1804 (syn. Chironomus thummi) by examining hereditary and somatic aberrations (mainly inversions) of the salivary gland polytene chromosomes. In total, 77 different types of inherited inversion sequences and 184 different types of somatic inversions were found. The median percent frequency of inherited inversions was 1.4% and karyotypic divergence between populations was very low. Most hereditary inversions were endemic and always in a heterozygous state. Only six inversion sequences, each of them shared by two very distant populations, may be considered a relic of very ancient ancestral inversions. Unlike inherited inversions, occurrence of somatic aberrations seems to increase with the overall rise in the level of heavy metal pollution of the sediments from which larvae were sampled. In contrast with what occurs in populations of other chironomid species, populations of C. riparius do not seem to undergo a process of cytogenetic differentiation.     

Karyotype of the midge Chironomus heterodentatus Konstantinov from the group Obtusidens (Diptera, Chironomidae)

The karyotype of Chironomus heterodentatus, belonging to the obtusidens-group described by Konstantinov from the Volga in 1956, was studied in detail. Combinations of chromosomal arms are AB, CD, EF and G (cytocomplex thummi). The Ch. heterodentatus karyotype cleary differs from those of other members of the obtusidens-group with a species-specific banding pattern in arms A, B, C and D. Chromosomal polymorphism on homo- and heterozygous inversions was found in arms, A, B, D, E and G. 19 inversion banding sequences and their 27 genotypic combinations have been recorded. The shortest arm G is highly polymorphic. Heterozygotes on the Balbiani ring activity were found in arm G along with homo- and heterozygotes on inversions. B-chromosomes with a frequency equal to 2.7-25.0% were recorded in some Volga populations.     

Insertional polymorphism of a non-LTR mobile element (NLRCth1) in European populations of Chironomus riparius (Diptera, Chironomidae) as detected by transposon insertion display.

The midge Chironomus riparius is distributed all over the Palearctic region and is well characterized both at the morphological and cytogenetic levels. Here we describe a population study based on the insertional polymorphism of the retroposon NLRCth1, by means of a S-SAP (sequence-specific amplification polymorphism) derived technique (transposon insertion display; TID). While a previous study of allozyme polymorphism in Russian samples showed little variability, all the amplicons we identified are polymorphic. Genetic distances between 6 natural populations were calculated according to Nei and did not show a positive correlation with geographic distances. The genetic diversity detected among individuals of a given population was one order of magnitude higher than that among populations. However, the value of phi(ST) was significant (p < 0.001) and indicates that natural populations are more genetically differentiated than random samples of individuals.     

Cytogenetic characteristics of a population of Chironomus riparius Meigen 1804 (Diptera, Chironomidae) from a polluted Po river station

A population of Chironomus riparius from a Po river station near Moncalieri (a trace-metal polluted station) was studied. In this population was established a great variability of band structure of polytene chromosomes as well as paracentric heterozygous inversions, deletions, deficiencies, partial breaks, diploid chromosome fragments, and changes in functional activity and appearance of heterochromatin. In arms A through F, some bands had an increased size compared to the standard chromosomic map. Some bands appeared in a heterozygous or normal homozygous state or were amplified. In all arms, many condensed stable bands appeared in the decondensed state when compared to the standard map. Asynaptic zones in arms E and G as well as heterozygous Balbiani rings and NORs were established. Very often the 4th chromosome was almost completely heteropycnotic and looded like a pompon chromosome. For the first time in this species, a high frequency of ectopic pairings of different arms was observed. Telomeric regions involved in ectopic pairings had a granular appearance, as did some centromeres. The hypothesis is advanced that such a high frequency of structural rearrangements could be correlated with genomic distribution of specific mobile elements.     

Gytogenetics of Chironomus riparius L. from the fishpond in Borok village (Diptera, Chironomidae)

We have found three inherited inversions in Chironomus riparius populations from the Borok fishpond, namely: (A3d-B1a) in the arm A (C5a-C6a) in the arm D and (B3b-4d/e) in the arm F. Increase of heterochromatin in some bands of chromosome F (B3h, B3h + B3c--C1a) and puffs appearance in the arms C, D and E have been observed. We saw also changes in functional activity of nucleolar organizer (N) and Balbiani rings (BRe/BRb). It has been found that some of inversion breakpoints coincide with the Alu and Hinf satellite DNA localization sites.     

Polymorphism and divergence of multilocus DNA markers in sibling species Chironomus riparius Meigen and Chironomus piger Strenzke (Diptera, Chironomidae)

Intra- and interspecific variation and divergence of multilocus markers for genomic DNA of the sibling species from the thimmi group, Chironomus riparius and C. piger, were studied by PCR with arbitrary primers (RAPD). A high level of RAPD polymorphism was determined in both laboratory and natural populations of these species. The genetic distances were estimated between the C. riparius populations and between the sibling species C. riparius and C. piger. The genetic distance between C. riparius and C. piger was 4 to 5 times higher than that between the C. riparius populations. A comparison of the variation and divergence for the RAPD markers with those for other genomic markers--enzyme-coding genes and chromosomes (linked gene groups)--showed that different components of the genome differed in their contribution to the genome divergence.     

Ultrastructure of the retrocerebral complex in the adult midge,chironomus riparius MG (Diptera : Chironomidae)

The organisation of the retrocerebral complex of adult Chironomus riparius Mg. (Diptera : Chironomidae) has previously been compared with that of the larva. The primary objective of the present work was to examine the ultrastructure of the components of the complex in neoimaginal male C. riparius to enable their further comparison with similar structures in other dipterans and determine whether this information supports the contention that the midge has a relatively primitive neuroendocrine system.The hypocerebral ganglion has a complex innervation, is composed of non- neurosecretory neurons and glial cells and, with the adjacent aortic wall, is the major neurohaemal site of the complex. The corpora cardiaca are not intimately associated with the ganglion. They are not apparently neurohaemal but are composed of cells containing granules with a bimodal size distribution. The corpora allata are of the pseudolymphoid type and are devoid of extracellular stromal channels. Peritracheal tissue persists into the neoimaginal midge but is apparently inactive and undergoing degeneration.The post-cerebral glands have been included in all previous accounts of the retrocerebral complex in chironomids but the present work confirms the view that they are not endocrine in nature despite their close proximity to the corpora cardiaca. Their ultrastructural appearance suggests that they may be functionally related to pericardial cells in other insects.It is concluded that the retrocerebral complex is indeed rather primitive both in its organisation and in the structure of its components.     

Polymorphism and Divergence of Multilocus DNA Markers in Sibling Species Chironomus riparius Meigen and Chironomus piger Strenzke (Diptera,Chironomidae)

Intra- and interspecific variation and divergence of multilocus markers for genomic DNA of the sibling species from the thimmi group,Chironomus riparius and C. piger, were studied by PCR with arbitrary primers (RAPD). A high level of RAPD polymorphism was determined in both laboratory and natural populations of these species. The genetic distances were estimated between the C. riparius populations and between the sibling species C. riparius and C. piger. The genetic distance between C. riparius andC. piger was 4 to 5 times higher than that between the C. riparius populations. A comparison of the variation and divergence for the RAPD markers with those for other genomic markers—enzyme-coding genes and chromosomes (gene linkage groups)—showed that different components of the genome differed in their contribution to the genome divergence.     

Seasonal dynamics and larval drift of Chironomus riparius (Diptera) in a metal contaminated lowland river

Chironomus riparius is one of the insect species which inhabit polluted rivers in large densities, indicating a high adaptive capacity. Previous studies showed that this capacity is expressed by the occurrence of adapted strains in metal polluted rivers. Differences in life history between metal-exposed and non-exposed midges have been demonstrated in laboratory experiments, and therefore a comparative field study of seasonal dynamics was carried out at two metal polluted sites and one reference site. Just downstream from a massive metal discharge, seasonal dynamics were almost identical to the upstream reference site. Circa four generations per year were produced. Further downstream, lower larval densities were recorded, especially during the second half of the sampling period. The influx of upstream C. riparius larvae into polluted sites was estimated by measuring larval drift just upstream from the point source of metal contamination and indicated a massive input to the standing stock downstream. It is concluded that drift of non-tolerant larvae is dominating the seasonal dynamics of midges downstream. Accordingly, genetic uniformity of chironomids inhabiting upstream and downstream sites is expected most of the time. However, research performed during the last decade, demonstrated that genetically adapted strains of C. riparius may develop at certain stages in the seasonal cycle. However, a stable metal-adapted C. riparius population at the first downstream site, is most likely present on rare occasions only.     

Mechanical,sensory and glandular structures in the tarsal unguitractor apparatus of Chironomus riparius (Diptera,Chironomidae)

Summary The legs of chironomid midges from a laboratory colony were examined in the region of the joint between the fifth tarsal segment and the pretarsus, especially the surface of the unguitractor and the manner in which the unguitractor fits into a ventromedian groove in the edge of the tarsus when the joint is flexed. The region was reconstructed from serial sections to clarify the spatial relations of the internal structures to one another and to the external structures. Ultrastructural characteristics of the cells and cuticle suggest a secretory function of the unguitractor. An amphinematic scoloparium is suspended between the point at which the unguitractor attaches to its tendon and the transverse diaphragm within the tarsus. This mechanoreceptor could serve as a proprioceptive sensor of the position of unguitractor and tarsus; it could also be an exteroceptor, sensing vibration in the substrate and/or the air. In the context of functional morphology, the clamping of the unguitractor in the ventral hollow in the tarsus could have the effect of resetting the sensitivity of the sensor. On the other hand, this arrangement could also simply act as an energy-saving means of fixing the claws in the grasping position for long periods.Abbreviations aj adhering junction - bl basal labyrinth - bm basement membrane - cc cuticular cap - cd cellular diaphragm - cth cuticular thickening - dp matrix with dense particles - ec enveloping cells - f filaments - fot funnel-like opening of tendon - ic intracellular canaliculi - lp lateral process of ec - pAc process of A cell - pv pulvillus - r rootlets of cilia - sc scolopale cell - sco scoloparium - sd septate desmosomes - t tendon - tb tubular body - tu tubules - u unguis, or claw - upl unguitractor plate