Extraordinary Conservation of Cysteines Among Homologous Chironomus Silk Proteins sp185 and sp220

Aquatic larvae of the midge, Chironomus tentans, synthesize a 185-kDa silk protein (sp185) with the cysteine-containing motif Cys-X-Cys-X-Cys (where X is any residue) every 20–28 residues. We report here the cloning and full-length sequence of cDNAs encoding homologous silk proteins from Chironomus pallidivittatus (sp185) and Chironomus thummi (sp220). Deduced amino acid sequences reveal proteins of nearly identical mass composed of 72 blocks of 20–28 residues, 61% of which can be described by the motif X_5–8-Cys-X₅-(Trp/Phe/Tyr)-X₄-Cys-X-Cys-X-Cys. Spatial arrangement of these residues is preserved more than surrounding sequences. cDNA clones enabled us to map the genes on polytene chromosomes and identify for the first time the homolog of the Camptochironomus Balbiani ring 3 locus in Chironomus thummi. The apparent molecular weight difference between these proteins (185 vs 220 kDa) is not attributable to primary structure and may be due to differential N-linked glycosylation. DNA distances and codon substitutions indicate that the C. tentans and C. pallidivittatus genes are more related to each other than either is to C. thummi; however, substitution rates for the 5′- and 3′-halves of these genes are different. Blockwise sequence comparisons suggest intragenic variation in that some regions evolved slower or faster than the mean and may have been subjected to different selective pressures. Received: 30 August 1996 / Accepted: 6 November 1996     

Subsidies for production of Chironomus calligraphus larvae (Chironomidae,Diptera) in the laboratory

The objective of this study was to evaluate the effect of temperature, type of mineral substrate, and type and amount of feed on the growth of Chironomus calligraphus as a subsidy for the production of Chironomus larvae to feed the fish. Egg production was evaluated at 22, 25, and 28?°C on fine (0.074–0.210?mm), or silty sand (0.002–0.250?mm), and no mineral substrate as control. For larval production, 0.10, 0.20, and 0.30?mg/larvae of fish feed, poultry feed, or turkey manure were used as food. The highest number of egg masses was produced when mineral substrates and 25?°C were used, but the higher number of eggs/mass was registered in the control. Therefore, the total number of eggs and hatched larvae produced was the same in the presence or in the absence of mineral substrates (p?>?0.05). The best results for survival and growth were recorded for larvae reared with fish or poultry food. The increase in the amount of feed was correlated with weight gain only for larvae fed with poultry feed, with no reduction in survival.     

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.     

Intraspecific and interspecific variation in drumming signals in the genus Capnioneura (Plecoptera: Capniidae)

The male drumming calls of four species of the genus Capnioneura, and for the first time, the female answer signal from one species of this genus are described. Only the male call of C. mitis from a southern Iberian Peninsula is known. In the current study, the male calls of three additional C. mitis populations from two different mountain drainages and the male calls of C. gelesae, C. libera and C. petitpierreae are described and analysed. The calls were near-ancestral and percussive signals with variable numbers of beats and approximately constant, monophasic beat intervals. The mean interbeat intervals were species specific, ranging from 250 to 1500 ms. The C. mitis female answer interbeat intervals were similar to the call but composed of 3–4 beats. Intraspecific and interspecific variation is discussed and these results support the hypothesis that stonefly drumming plays an important role as a reproductive isolating mechanism.     

Comparison of Chironomus usenicus and Chironomus curabilis with species of the group plumosus (Diptera) inferred from the mitochondrial DNA Gene COI and by the polytene chromosomes banding pattern

The sequence of a 595-bp fragment of the mitochondrial COI gene was determined for the species Chironomus usenicus and Chironomus curabilis of the genus Chironomus. Phylogenetic reconstructions based on the analysis of the COI gene sequence coincide on the whole with cytogenetic data, permitting Ch. usenicus and Ch. curabilis to be regarded as members of the group plumosus. Chironomus usenicus and Ch. plumosus have identical COI gene sequences. Two hypotheses explaining this identity are considered: inheritance of mtDNA from one of the parental species during hybridogenesis and horizontal transfer of mitochondrial genes.     

C-banding in the polytene chromosomes of species of the plumosus group (Diptera,Chironomidae) and their experimental hybrids

The localization and amount of heterochromatin in the plumosus group were studied, including the species Chironomus plumosus L., C. vancouveri and C. balatonicus. The appearance of C bands of Chironomus plumosus in several European populations is traced. The role of the C heterochromatin in the differentiation of this species is discussed. From the evolutionary point of view the Swiss populations, in which large centromere heterochromatin blocks have been discovered, are more varied as to the amount of heterochromatin. The importance of duplications for this process is pointed out. The chromosomes of the individuals from C. vancouveri and C. balatonicus have centromeric, telomeric and interstitial heterochromatin. The centromeric heterochromatin is represented by thin C-bands. The particularities in the appearance of C heterochromatin in C. vancouveri and C. balatonicus reflect the structural peculiarities of their chromosomes. The change in the euchromatin regions in these forms is discussed in the light of transformation of euchromatin to heterochromatin in the process of evolution.The appearance of heterochromatin in hybrids (between populations and between species) created experimentally is traced. A change has been discovered in the appearance of heterochromatin in the hybrids compared to the initial parent forms. This difference is expressed more strongly in inter-species hybrids than in interpopulation hybrids of C. plumosus.     

On the Possibility of Hybridogenesis in the Origin of Midge Chironomus usenicus Loginova et Beljanina (Chironomidae,Diptera)

Hybridogenesis as a possible way of speciation in Chironomidae was considered with special reference to the species Chironomus usenicus resulting from hybridization between C. plumosus and C. behningi. The three species had 2n = 8 and belonged to the thummi cytocomplex with chromosome arm combinations AB, CD, EF, and G. Arm G had a marker chromosome disk sequence (CDS) and was used to demonstrate the hybrid origin of C. usenicus. Most C. usenicus larvae were heterozygous in CDS of arm G. CDS use G2 proved to be identical to CDS beh G1 ofC. behningiand CDS use G1, to CDS plu G1 of C. plumosus. It was assumed that C. usenicus results from hybridization between eurybiont C. plumosus and stenobiont C. behnigni at the boundary of their species areas, in freshwater or brackish water bodies of the southern Saratov oblast and northern Kazakhstan. Morphologically and karyotypically, the hybrid was probably similar to C. plumosus. Crosses with C. plumosus eliminated virtually all C. behningi chromosome sequences from the karyotype of the hybrid. Further chromosome divergence resulting in C. usenicus involved a number of chromosome rearrangements, including duplication of pericentric heterochromatin and other chromosome regions; inversion, which occurred in arm F (regions 13–16) and was fixed in the karyotype; and other paracentric inversions and deletions accumulated in heterozygote in the karyotype pool of the species. Since C. behningi was eliminated from the introgression zone and its species area reduced, the assimilation character was assumed for introgressive hybridization of C. behningi and C. plumosus.     

Differences between sympatric populations of <Emphasis Type="Italic">Eotetranychus carpini</Emphasis> collected from <Emphasis Type="Italic">Vitis vinifera</Emphasis> and <Emphasis Type="Italic">Carpinus betulus</Emphasis>: insights from host-switch experiments and molecular data

Eotetranychus carpini (Oudemans) is an important pest of grapevine (Vitis vinifera L.) in southern Europe. This mite is also found on a number of different plants, including Carpinus betulus L., which commonly occurs in stands and hedgerows bordering vineyards, where it may serve as a potential mite reservoir. The economic importance of this pest has motivated a number of studies aimed at investigating whether the mites found on V. vinifera and C. betulus are conspecific. The results obtained to date have been inconclusive. In this study, we used biological and molecular approaches to investigate this issue. First, we conducted host-switch experiments to test the ability of E. carpini to develop on an alternative host plant, using mite populations originally collected on either C. betulus or V. vinifera plants from the same area. Second, we investigated DNA-based differentiation using nucleotide sequences of the ITS1-5.8S-ITS2 region of the ribosomal DNA of individual E. carpini from the populations examined in our host-plant experiments. We also analyzed sequences of individuals collected in other regions (Italy and Slovenia) to estimate species variation. The results from our host-switch experiments suggest the differentiation of mites collected on the two hosts. Mites collected from C. betulus did not survive and reproduce on V. vinifera and vice versa. Our molecular work revealed significant genetic differentiation between the mites collected from the two hosts, but no evidence of genetic variation among specimens collected from the same host species. Our results indicate the existence of host races of E. carpini.     

Conservation and variability of sequence-tagged microsatellite sites (STMSs) from chickpea (Cicer aerietinum L.) within the genus Cicer

The conservation of 90 microsatellite-flanking sequences from chickpea in 39 accessions of eight annual and 1 accession of a perennial species of the genus Cicer was investigated. All of the primer sequences successfully amplified microsatellites in related species, indicating the conservation of microsatellite-flanking sequences in chickpea’s relatives. However, the degree of conservation of the primer sites varied between species depending on their known phylogenetic relationship to chickpea, ranging from 92.2% in C. reticulatum, chickpea’s closest relative and potential ancestor, down to 50% for C. cuneatum. A phylogenetic tree revealed that chickpea and the other members of its crossability group were more closely related to the perennial C. anatolicum than to other annual species of the genus. Considerable variation in size and number of amplification products between and within species was observed. Sequence analysis of highly divergent amplification products proved that variation is either due to large differences in the number of microsatellite repeats or to the amplification of a locus unrelated to the one amplified from chickpea. Sequence information and bootstrapping using PAUP suggested that STMSs derived from chickpea may be efficiently and reliably used for synteny studies in chickpea’s crossability group, including C. anatolicum. However, care should be taken when applying these markers to other species of the genus. Considering the data presented here and the known historical record, the age of section Monocicer, including chickpea, is estimated to be about 100,000 years. Received: 13 August 1999 / Accepted: 11 November 1999     

The organization of the ribosomal RNA genes of Chironomus tentans and some closely related species

Southern gel analysis of total DNA from Chironomus tentans showed that the rRNA genes (rDNA) are homogeneous in structure. After cloning in Escherichia coli plasmid pBR313, the rDNA organisation was further studied by restriction fragment analysis and R-loop mapping. No heterogeneity could be detected by heteroduplex analysis of six different cloned rRNA cistrons. R-loop sizes of 1.69 and 3.63 kilobases (kb) were measured for the 18S and 28S rRNA coding sequences. The two spacers are 0.75 and 1.77 kb long. Southern gel analysis showed also a homogeneous rDNA structure for a Canadian population of C. tentans and C. pallidivittatus. The same technique indicated, however, that the rDNA of two other closely related species of C. thummi and C. melanotus is heterogeneous in structure. A possible correlation between this heterogeneity and the presence of heterochromatin in these species is discussed.     

Obba and Sebipora, new polypore genera related to Cinereomyces and Gelatoporia (Polyporales, Basidiomycota)

Cinereomyces clade is a newly proposed monophyletic group of polypores containing currently four genera and five species, including two promising biopulping fungi, Ceriporiopsis rivulosa and C. subvermispora. The Cinereomyces clade is well-delimited in nrDNA-based phylogenetic analysis, but its position in Polyporales remains unclear. Its closest relative may be found in the core polyporoid clade. Only a few morphological characters are common for all the species in the clade, e.g. CB– and CRB+ hyphae, white fruiting bodies, presence of oil, and middle-sized spores. Culturally, the species are unified by producing simple-septate generative hyhpae in the margin, which produce simple-clamped hyphae backwards. The genus Gelatoporia is the correct place for Ceriporiopsis subvermispora. Two new genera are described in the group: Obba to incorporate C. rivulosa and a new austral species, Obba valdiviana, known from southern Argentina and recorded here also from Tasmania, and Sebipora to accommodate a new species from tropical Asia, S. aquosa. ITS sequences imply that Eurasian Gelatoporia subvermispora may belong to a different species from the North American one. G. subvermispora is recorded as new to Indonesia.     

Phylogenetic placement and population structure of Indo‐Pacific bottlenose dolphins (Tursiops aduncus) off Zanzibar,Tanzania, based on mtDNA sequences

Phylogenetic placement of bottlenose dolphins from Zanzibar, East Africa and putative population differentiation between animals found off southern and northern Zanzibar were examined using variation in mtDNA control region sequences. Samples (n= 45) from animals bycaught in fishing gear and skin biopsies collected during boat surveys were compared to published sequences (n= 173) of Indo‐Pacific bottlenose dolphin, Tursiops aduncus, from southeast Australian waters, Chinese/Indonesian waters, and South African waters (which recently was proposed as a new species) and to published sequences of common bottlenose dolphin, Tursiops truncatus. Bayesian and maximum parsimony analyses indicated a close relationship between Zanzibar and South African haplotypes, which are differentiated from both Chinese/Indonesian and Australian T. aduncus haplotypes. Our results suggest that the dolphins found off Zanzibar should be classified as T. aduncus alongside the South African animals. Further, analyses of genetic differentiation showed significant separation between the T. aduncus found off northern and southern Zanzibar despite the relatively short distance (approximately 80 km) between these areas. Much less differentiation was found between southern Zanzibar and South Africa, suggesting a more recent common evolutionary history for these populations than for the northern and southern Zanzibar populations.     

Phylogenetic Relationships among Holarctic Populations of Chironomus entis and Chironomus plumosus in View of Possible Horisontal Transfer of Mitochondrial Genes

In eight Holarctic populations of two typical chironomid sibling species of the plumosus group, Chironomus entisandChironomus plumosus, nucleotide sequences of mitochondrial (cytb) and nuclear (gb2b) gene regions were examined. The phylogenetic trees reflecting the evolutionary histories of the nuclear and mitochondrial markers exhibited significant differences. On the tree based on the nuclear gene sequences the populations clustered according to their species affiliation, whereas on the tree based on the mitochondrial gene sequences the populations were grouped according to their geographic position. This discrepancy is probably explained by mitochondrial gene flow between sympatric species with incomplete reproductive isolation (sibling species). Based on our results together with the earlier data on nuclear and mitochondrial gene sequences of some other species from the phylogenetic group plumosus, a scheme of phylogenetic relationships within this group is proposed. This scheme is in many ways different from the traditional view on the evolutionary relationships among species of the plumosus group.     

Spatio-temporal effects of selected physico-chemical variables of water, algae and sediment chemistry on the larval community of nuisance Chironomidae (Diptera) in a natural and a man-made lake in central Florida

Spatio-temporal distributions of Chironomus crassicaudatus, Chironomus decorus, Glyptotendipes paripes, Cryptochironomus fulvus and Tanypodinae midge larvae and selected associated environmental variables (physico-chemical variables of water, sediment chemistry and algal composition) were studied for one year in natural Lake Monroe and man-made Konomac Lake in central Florida. Ordination analysis (CCA with variation partitioning) showed that in each lake, sediment variables were the most important environmental variables. However, the most important proportion of species variability in pooled data from both lakes was explained by lake-specific combinations of water, sediment and algal variables. Chironomid larvae were ordinated with increasing depth as follows: G. paripes, C. fulvus, C. crassicaudatus, Tanypodinae, and C. decorus. This trend was similar for both lakes individually as well as for the pooled data. As water depth increased, organic matter content and associated metals concentrations in sediment increased. Glyptotendipes paripes displayed significant, negative correlation with water depth and sediment total carbon content, while C. decorus showed a significant positive correlation with these variables; C. crassicaudatus did not show any significant correlation with either of these variables. The algal composition in the pooled data from both lakes was another important factor affecting the larval distributions. The proportion of G. paripes and C. decorus larvae increased with filamentous algal density, and C. crassicaudatus larvae with increasing Cyanobacteria. Although both study lakes were very shallow, the effect of water depth (alone or shared with environmental variables) on chironomid larval community composition was substantially higher than the seasonality (sampling month) effect.     

Individual variations in the content of giant secretory polypeptides in salivary glands of Chironomus

Salivary glands in aquatic larvae of Chironomus are responsible for formation of a fiber that larvae use to construct feeding tubes. Major constituents of this fiber include a family (the sp-I family) of high M _r (1 × 10⁶) secretory polypeptides. Because of our interest in the polypeptide composition and polymerization of the salivary fiber we conducted a survey of the electrophoretic pattern of sp-I components found in salivary glands obtained from individual larvae. The survey encompassed ten strains of Chironomus tentans, three strains of Chironomus pallidivittatus and four strains of Chironomus thummi. Salivary glands from C. tentans and C. pallidivittatus contained at least four sp-I components (sp-Ia, sp-Ib, sp-Ic and sp-Id) that behave identically with regard to their electrophoretic mobility and detectability when larvae were exposed to galactose or glycerol. Sp-I components in C. thummi were generally fewer and not directly comparable by electrophoretic mobility to sp-I components in the other two species. During this survey two important alterations were observed in the electrophoretic pattern of sp-I components obtained from C. tentans and C. pallidivittatus. First, all four sp-I components exhibited, with a low frequency, double bands that appeared as slow-versus-fast electrophoretic variants of a particular component. Secondly, the relative steady-state level of each sp-I component fluctuated in comparison to other sp-I components in the same extract. This fluctuation varied such that any one sp-I component might appear as a single prominent component. Sp-I components are encoded by a multigene family located in Balbiani rings (BRs). Results presented here, in conjunction with known nucleotide sequence data from BR genes, led us to the following conclusions. The slow and fast electrophoretic variants observed for each sp-I component suggest that each corresponding BR gene may be able to expand and/or contract in size. The observed degree of independent fluctuation in the steady-state level of each sp-I component suggests that each BR gene may be able to regulate its expression independently from the others. Finally, the observation that salivary glands sometimes contained only one prominent sp-I component led us to hypothesize that, whereas salivary fibers might typically be heteropolymers comprised of two or more types of sp-I components, homopolymers comprised of only one sp-I component may also exist.     

Intercontinental karyotypic differentiation of Chironomus entis Shobanov, a Holarctic member of the C. plumosus group (Diptera, Chironomidae).

Analysis of banding sequences of polytene chromosomes in Palearctic (Russian) and Nearctic (North American) Chironomus entis shows strong karyotype divergence between populations on the two continents. Four out of seven chromosomal arms in the North American C. entis karyotype are characterized by sequences found only in the Nearctic. In total, 44 banding sequences are now known for this species across the Holarctic, including 22 exclusively Palearctic, 6 Holarctic, and 16 exclusively Nearctic sequences. The degree of cytogenetic differentiation between Palearctic and Nearctic C. entis populations is an order of magnitude greater than differentiation among populations within either continent, but is only one third as great as the cytogenetic distance between the sibling species C. entis and C. plumosus. C. entis is the only sibling species of C. plumosus uncovered during cytological identification of Chironomus species from more than 50 North American lakes, indicating that the plumosus sibling-species group is much smaller in the Nearctic than in the Palearctic, where a dozen sibling species are known. Cytogenetic distance values calculated between Nearctic and Palearctic representatives of both C. entis and its sibling species C. plumosus are similar, but result from different patterns of karyotype divergence. New World C. entis is distinguished from Old World populations by the 16 uniquely Nearctic sequences, four of which occur in the homozygous state. In contrast, North American C. plumosus has fewer uniquely Nearctic sequences, and only one that occurs as a homozygote. However, four chromosomal arms in C. plumosus that are polymorphic in the Palearctic show fixation, or near fixation, of Holarctic sequences in the Nearctic C. plumosus karyotype. Thus, both the fixation of Holarctic sequences, and the occurrence or fixation of distinctly Nearctic sequences, contribute significantly to karyotype divergence. Patterns of karyotype divergence in Palearctic and Nearctic populations of different Holarctic chironomid species are discussed relative to intercontinental cytogenetic differentiation in other dipterans.     

A FORENSIC AND PHYLOGENETIC SURVEY OF CAULERPA SPECIES (CAULERPALES,CHLOROPHYTA) FROM THE FLORIDA COAST,LOCAL AQUARIUM SHOPS,AND E‐COMMERCE: ESTABLISHING A PROACTIVE BASELINE FOR EARLY DETECTION1

Baseline genotypes were established for 256 individuals of Caulerpa collected from 27 field locations in Florida (including the Keys), the Bahamas, US Virgin Islands, and Honduras, nearly doubling the number of available GenBank sequences. On the basis of sequences from the nuclear rDNA‐ITS 1+2 and the chloroplast tufA regions, the phylogeny of Caulerpa was reassessed and the presence of invasive strains was determined. Surveys in central Florida and southern California of >100 saltwater aquarium shops and 90 internet sites revealed that >50% sold Caulerpa. Of the 14 Caulerpa species encountered, Caulerpa racemosa was the most common, followed by Caulerpa sertularioides, Caulerpa prolifera, Caulerpa mexicana, and Caulerpa serrulata. None of the >180 field‐collected individuals (representing 13 species) was the invasive strain of Caulerpa taxifolia or C. racemosa. With one exception (a sample of C. racemosa from a shop in southern California belonged to the invasive Clade III strain), no invasive strains were found in saltwater aquarium stores in Florida or on any of the internet sites. Although these results are encouraging, we recommend a ban on the sale of all Caulerpa species (including “live rock”) because: morphological identification of Caulerpa species is unreliable (>12% misidentification rate) and invasive strains can only be identified by their aligned DNA sequences, and because the potential capacity for invasive behavior in other Caulerpa species is far from clear. The addition of the Florida region to the genetic data base for Caulerpa provides a valuable proactive resource for invasion biologists as well as researchers interested in the evolution and speciation of Caulerpa.