The genomic basis of circadian and circalunar timing adaptations in a midge.

Organisms use endogenous clocks to anticipate regular environmental cycles, such as days and tides. Natural variants resulting in differently timed behaviour or physiology, known as chronotypes in humans, have not been well characterized at the molecular level. We sequenced the genome of Clunio marinus, a marine midge whose reproduction is timed by circadian and circalunar clocks. Midges from different locations show strain-specific genetic timing adaptations. We examined genetic variation in five C. marinus strains from different locations and mapped quantitative trait loci for circalunar and circadian chronotypes. The region most strongly associated with circadian chronotypes generates strain-specific differences in the abundance of calcium/calmodulin-dependent kinase II.1 (CaMKII.1) splice variants. As equivalent variants were shown to alter CaMKII activity in Drosophila melanogaster, and C. marinus (Cma)-CaMKII.1 increases the transcriptional activity of the dimer of the circadian proteins Cma-CLOCK and Cma-CYCLE, we suggest that modulation of alternative splicing is a mechanism for natural adaptation in circadian timing.

The importance of DNA barcode choice in biogeographic analyses - a case study on marine midges of the genus Clunio.

DNA barcodes are widely used for species identification and biogeographic studies. Here, we compare the use of full mitochondrial genomes versus DNA barcodes and other mitochondrial DNA fragments for biogeographic and ecological analyses. Our dataset comprised 120 mitochondrial genomes from the genus Clunio (Diptera: Chironomidae), comprising five populations from two closely related species (Clunio marinus and Clunio balticus) and three ecotypes. We extracted cytochrome oxidase c subunit I (COI) barcodes and partitioned the mitochondrial genomes into non-overlapping windows of 750 or 1500 bp. Haplotype networks and diversity indices were compared for these windows and full mitochondrial genomes (15.4 kb). Full mitochondrial genomes indicate complete geographic isolation between populations, but do not allow for conclusions on the separation of ecotypes or species. COI barcodes have comparatively few polymorphisms, ideal for species identification, but do not resolve geographic isolation. Many of the similarly sized 750 bp windows have higher nucleotide and haplotype diversity than COI barcodes, but still do not resolve biogeography. Only when increasing the window size to 1500 bp, two windows resolve biogeography reasonably well. Our results suggest that the design and use of DNA barcodes in biogeographic studies must be carefully evaluated for each investigated species.

Seasonally varying marine influences on the coastal ecosystem detected through molecular gut analysis.

Terrestrial predators on marine shores benefit from the inflow of organisms and matter from the marine ecosystem, often causing very high predator densities and indirectly affecting the abundance of other prey species on shores. This indirect effect may be particularly strong if predators shift diets between seasons. We therefore quantified the seasonal variation in diet of two wolf spider species that dominate the shoreline predator community, using molecular gut content analyses with general primers to detect the full prey range. Across the season, spider diets changed, with predominantly terrestrial prey from May until July and predominantly marine prey (mainly chironomids) from August until October. This pattern coincided with a change in the spider age and size structure, and prey abundance data and resource selection analyses suggest that the higher consumption of chironomids during autumn is due to an ontogenetic diet shift rather than to variation in prey abundance. The analyses suggested that small dipterans with a weak flight capacity, such as Chironomidae, Sphaeroceridae, Scatopsidae and Ephydridae, were overrepresented in the gut of small juvenile spiders during autumn, whereas larger, more robust prey, such as Lepidoptera, Anthomyidae and Dolichopodidae, were overrepresented in the diet of adult spiders during spring. The effect of the inflow may be that the survival and growth of juvenile spiders is higher in areas with high chironomid abundances, leading to higher densities of adult spiders and higher predation rates on the terrestrial prey next spring.

Aquatic insect communities in small stream in the south of Brazil.

Pollution of rivers and streams, by anthropic action, is characterized as an environmental, social, and sanitary problem. Factors such as the association between the marginal vegetation, the distribution of the substrates in the riverbed, and the availability of allochthonous organic matter influence the distribution and composition of the aquatic entomofauna. The objective of this study was to analyze the structure of aquatic insect communities in a pasture stream in northern Paraná, southern Brazil, with emphasis on the groups of indicators of good water quality, thus inferring the conditions of its preservation. Samples were collected from July to October in three parts of the stream (P1, P2, and P3), where the insect faune was collected with the aid of a sieve in the foliage substrate and washing of rocks and decomposing pieces of wood. A total of 1323 individuals were collected, being Chironomidae (Diptera) the most abundant taxon. The analysis of the biotic indices (EPT/Chironomidae, IBF, BMWP, and BMWP/ASPT) and diversity indicated better preservation conditions at points P1 and P3 where the riparian forest was well preserved, with less exposure to the stream bed. In P2, the entomofauna presented less diversity and the biotic indexes indicated loss of water quality, showing the impacts of changes in the marginal vegetation of this section. In a generally preserved aquatic environment, small changes in its vegetation are sufficient to cause an imbalance in the aquatic insect community, showing the efficiency of these organisms as bio-indicators and the sensitivity of biotic indexes.

DNA barcode data reveal biogeographic trends in Arctic non-biting midges.

Chironomid flies (non-biting midges) are among the most abundant and diverse animals in Arctic regions, but detailed analyses of species distributions and biogeographical patterns are hampered by challenging taxonomy and reliance on morphology for species-level identification. Here we take advantage of available DNA barcode data of Arctic Chironomidae in BOLD to analyse similarities in species distributions across a northern Nearctic - West Palearctic gradient. Using more than 260 000 barcodes representing 4666 BINs (Barcode Index Numbers) and 826 named species (some with interim names) from a combination of public and novel data, we show that the Greenland chironomid fauna shows affinities to both the Nearctic and the West Palearctic regions. While raw taxon counts indicate a strong Greenland - North American affinity, comparisons using Chao's dissimilarity metric support a slightly higher similarity between Greenland and West Palearctic chironomid communities. Results were relatively consistent across different definitions of species taxonomic units, including morphologically determined species, BINs, and superBINs based on a ∼4.5% threshold. While most taxa found in Greenland are shared with at least one other region, reflecting circum-Arctic dispersal, our results also reveal that Greenland harbours a small endemic biodiversity. Our exploratory study showcases how DNA barcoding efforts using standardized gene regions contribute to an understanding of broad-scale patterns in biogeography by enabling joint analysis of public DNA sequence data derived from diverse prior studies.

Rhabdostylid Ciliates (Ciliophora, Peritrichia, Epistylididae) as Epibionts on Chironomid Larvae: Evidence of High Specificity and Association with Organic Pollution.

Although epibiont ciliates make up a significant part of the biomass in aquatic ecosystems and may cause perceptible alterations in the population dynamics of their hosts, studies on the extrinsic and intrinsic factors that control the abundance of these microorganisms are scarce in literature. In the present study, we investigated the colonization site and intensity of rhabdostylid epibiont upon chironomid larvae and assessed the influence of organic pollution and chironomid communities on the prevalence and abundance of epibiont ciliates at five sampling stations (two in rural areas and three in urban areas) along a neotropical urban stream over a period of 12 months. Among the 24 genera of chironomids found and the 31,976 larvae analyzed, 96.12% belonged to the Chironomus genus, of which 16.95% (5212) were colonized by Rhabdostyla aff. chironomi. The infestation intensity varied from one to 67 individuals per host with an average intensity of 4.86 (± 33.45). Ciliates were only found colonizing the chironomids' ventral tubules. The high number of chironomid larvae, high host- and site-specificity, low infestation intensity, and absence of apparent structural damage to hosts evidence an intimate relationship between epibiont and basibiont as well as a possibly long coevolutionary history. Both prevalence and abundance of epibiont ciliates were correlated to the pollution rate of the studied stream. There was an alteration in the composition and structure of the chironomid community along the sampling stations. Numeric dominance of Chironomus-tolerant chironomids and its direct correlation to infestation prevalence and to ciliates abundance highlights the predilection of both peritrich ciliates and Chironomus larvae for organically-enriched environments.

Morphological identification and COI barcodes of adult flies help determine species identities of chironomid larvae (Diptera, Chironomidae).

Establishing reliable methods for the identification of benthic chironomid communities is important due to their significant contribution to biomass, ecology and the aquatic food web. Immature larval specimens are more difficult to identify to species level by traditional morphological methods than their fully developed adult counterparts, and few keys are available to identify the larval species. In order to develop molecular criteria to identify species of chironomid larvae, larval and adult chironomids from Western Lake Erie were subjected to both molecular and morphological taxonomic analysis. Mitochondrial cytochrome c oxidase I (COI) barcode sequences of 33 adults that were identified to species level by morphological methods were grouped with COI sequences of 189 larvae in a neighbor-joining taxon-ID tree. Most of these larvae could be identified only to genus level by morphological taxonomy (only 22 of the 189 sequenced larvae could be identified to species level). The taxon-ID tree of larval sequences had 45 operational taxonomic units (OTUs, defined as clusters with >97% identity or individual sequences differing from nearest neighbors by >3%; supported by analysis of all larval pairwise differences), of which seven could be identified to species or 'species group' level by larval morphology. Reference sequences from the GenBank and BOLD databases assigned six larval OTUs with presumptive species level identifications and confirmed one previously assigned species level identification. Sequences from morphologically identified adults in the present study grouped with and further classified the identity of 13 larval OTUs. The use of morphological identification and subsequent DNA barcoding of adult chironomids proved to be beneficial in revealing possible species level identifications of larval specimens. Sequence data from this study also contribute to currently inadequate public databases relevant to the Great Lakes region, while the neighbor-joining analysis reported here describes the application and confirmation of a useful tool that can accelerate identification and bioassessment of chironomid communities.

A contribution to the systematics of Australasian Tanytarsini (Diptera: Chironomidae): first descriptions from New Caledonia.

First specific records of chironomids of the tribe Tanytarsini from New Caledonia based on detailed descriptions of new species are presented. Cladotanytarsus (Cladotanytarsus) stylifer sp. nov. and its closest relatives, i.a. Cladotanytarsus (C.) isigacedeus (Sasa et Suzuki, 2000), comb. nov., known from males bearing extraordinarily elongate hypopygial anal points are diagnosed. Paratanytarsus mirificus sp. nov. is described as adult male with unique structure of its hypopygium and shortened antennae. Diagnostic description of Tanytarsus fuscithorax Skuse, 1889 is also complemented.

A review of the genus Parorthocladius Thienemann, 1935 (Diptera: Chironomidae: Orthocladiinae) from the Russian Far East.

Three new species of the genus Parorthocladius Thienemann, P. lazovskiensis sp. nov., P. plolabius sp. nov., and P. tyurkini sp. nov. from the Russian Far East are described and figured. Descriptions of pupae and larvae of one indeterminate species, Parorthocladius sp. 1, are also presented. Keys to determination of adult males, pupae and fourth instar larvae of Far-Eastern species Parorthocladius are provided.

Nomenclatural corrections to Australian species of Cricotopus (Wulp) (Diptera; Chironomidae).

Our attention has been drawn to lapsi and errors in a recent publication in this journal concerning Cricotopus Wulp (Diptera: Chironomidae) (Drayson et al., 2015).

A new record of Chironomus (Chironomus) acidophilus Keyl (Diptera, Chironomidae) from the Uzon volcanic caldera (Kronotsky Reserve, Kamchatka Peninsula, Russia), its karyotype, ecology and biology.

Morphology, cytology, ecology and biology of Holarctic Chironomus (Chironomus) acidophilus Keyl, 1960 (Diptera, Chironomidae) was examined from material collected in the geothermal Vosmerka Lake (pH=2.0-2.5). An illustrated redescription of C. acidophilus is given on the basis of adult males reared from field-collected pupae, and of simultaneously collected larvae. Additional larvae belonging to the pseudothummi-complex were identified as C. acidophilus on the basis of their karyotype. The karyotype of C. acidophilus (2n=8) and detailed mapping of the 4 chromosome arms A, E, D and F are provided. The population of C. acidophilus from Kamchatka was found to be karyologically monomorphic. Information on distribution and ecology of C. acidophilus from Vosmerka Lake (total mineralization 1583.5 mg/l) is also given. Chironomus acidophilus is the only species of aquatic insects recorded in this lake. Lack of competition and a richness of food resources contribute to the high abundance (35161 ind./m2) and biomass (11.342 g/m2) of the larvae of C. acidophilus in Vosmerka Lake.

A rapid method of species identification of wild chironomids (Diptera: Chironomidae) via electrophoresis of hemoglobin proteins in sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE).

Studying aquatic benthic macroinvertebrates (BMIs) in the field requires accurate taxonomic identification, which can be difficult and time consuming. Conventionally, head capsule morphology has been used to identify wild larvae of Chironomidae. However, due to the number of species and possible damage and/or deformity of their head capsules, another supporting approach for identification is needed. Here, we provide hemoglobin (Hb) protein in hemolymph of chironomids as a new biomarker that may help resolve some of the ambiguities and difficulties encountered during taxonomic identification. Chironomids collected from two locations in Maine and New Jersey, USA were identified to the genus level and in some cases to the species-level using head capsule and body morphologies. The head capsule for a particular individual was then associated with a corresponding Hb protein profile generated from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Distinct Hb profiles were observed from one group (Thienemannimyia) and four genera (Chironomus, Cricotopus, Dicrotendipes, and Glyptotendipes) of chironomids. Several species were polymorphic, having more than one Hb profile and/or having bands of the same size as those of other species. However, major bands and the combination of bands could distinguish individuals at the genus and sometimes species-level. Overall, this study showed that Hb profiles can be used in combination with head capsule morphology to identify wild chironomids.

Integrating DNA barcodes and morphology for species delimitation in the Corynoneura group (Diptera: Chironomidae: Orthocladiinae).

In this study, we use DNA barcodes for species delimitation to solve taxonomic conflicts in 86 specimens of 14 species belonging to the Corynoneura group (Diptera: Chironomidae: Orthocladiinae), from the Atlantic Forest, Brazil. Molecular analysis of cytochrome c-oxidase subunit I (COI) gene sequences supported 14 cohesive species groups, of which two similar groups were subsequently associated with morphological variation at the pupal stage. Eleven species previously described based on morphological criteria were linked to DNA markers. Furthermore, there is the possibility that there may be cryptic species within the Corynoneura group, since one group of species presented internal grouping, although no morphological divergence was observed. Our results support DNA-barcoding as an excellent tool for species delimitation in groups where taxonomy by means of morphology is difficult or even impossible.

A new genus of Podonominae (Diptera: Chironomidae) in Late Eocene Rovno amber from Ukraine.

The genus Palaeoboreochlus Baranov et Andersen, n. gen. is erected based on P. inornatus Baranov et Andersen, n. sp. described from a male found in Late Eocene Rovno amber. The new genus groups with Boreochlus Edwards in the tribe "Boreochlini".

Species of the subgenus Chaetolabis, townes 1945 of the genus Chironomus, Meigen 1803 (Diptera: Chironomidae), from a cytogenetic perspective.

The polytene chromosomes of three North American species in the subgenus Chaetolabis of the genus Chironomus are described and compared to the published karyotypes of two Palearctic species. Chironomus (Chaetolabis) atroviridis has four polytene chromosomes, like the Palearctic C. globulus, while C. ochreatus and the North American populations of C. bitumineus each have three chromosomes due to tandem fusion of arm G to arm E, as in the Palearctic populations of C. bitumineus. The results support the existence of distinct species, particularly clarifying that the names C. atroviridis, C. bitumineus and C. ochreatus cannot be synonyms, as was suggested by some previous workers. The question of whether Lobochironomus "sp. Ya6" might be conspecific with C. macani is also discussed.

A new species of Bilyjomyia Niitsuma et Watson from Japan, with keys to species of the genus (Diptera: Chironomidae).

Descriptions of the male, female imagos, pupa and larva are given for Bilyjomyia parallela n. sp. collected from Japan. This is the third known species belonging to the genus Bilyjomyia. Keys to species of the genus are provided.

On taxonomy of Hydrobaenus fries, 1830 (Diptera: Chironomidae: Orthocladiinae) from the Russian Far East, with a key to species.

Additions and corrections to the diagnosis of the genus Hydrobaenus, a redescription for adult male and immature stages of H. distinctus (Makarchenko et Makarchenko) comb. n. , a taxonomic notes to H. jacuticus Makarchenko et Makarchenko and identification keys of adult males, pupae and larvae of Hydrobaenus species from the Russian Far East are given.

A new species of sponge-dwelling Oukuriella (Chironomidae) from Brazil.

Oukuriella Epler, 1986 is an exclusively Neotropical genus that comprises two well supported clades: inhabitants of freshwater sponges and inhabitants of immersed wood (saproxylophilic). In this work, males of a new chironomid species, Oukuriella froehlichi sp. n., are described and illustrated. Specimens were collected near stream in the Brazilian Atlantic Forest. Phylogenetic analyses performed in this study show that the newly discovered species belongs in the clade of freshwater sponge inhabitants.

To systematics of the genus Saetheria Jackson (Diptera, Chironomidae) from the Russian Far East.

The genus Saetheria Jackson from the Russian Far East is reviewed. The males of S. reissi Jackson, 1977, S. tamanipparai (Sasa, 1983) and S. tylus (Townes, 1945) are redescribed and figured. The pupa of S. reissi is redescribed and illustrated. The larva of S. reissi Jackson is described for the first time. Comments on the systematics and distribution of each species are provided. Paracladopelma kisopediformis Sasa, Kondo, 1993 is designated a new junior synonym of S. reissi Jackson, 1977. Keys to the males, pupae and larvae of the Russian Saetheria are given.

A taxonomic revision of genus Labrundinia Fittkau, 1962 (Diptera: Chironomidae: Tanypodinae).

The species of the genus Labrundinia (Diptera: Chironomidae: Tanypodinae: Pentaneurini) are revised, described and figured. Keys to known adult males, pupae and larvae are provided. Fourteen previously known species are redescribed, and 25 species from Neotropical region are diagnosed and described as new to science. These species treated are L. amandae sp. n., L. amazonica sp. n., L. becki Beck et Beck, L. carolae sp. n., L. costaricae sp. n., L. fera Roback, L. fiorelinii sp. n., L. fittkaui sp. n., L. fosteri Roback, L. hirsuta Roback, L. gregi sp. n., L. jasoni sp. n., L. johannseni Beck et Beck, L. kogilamae sp. n., L. longipalpis Goetghebuer, L. mayaca sp. n., L. meta Roback, L. multidentata sp. n., L. nathani sp. n., L. neopilosella Beck et Beck, L. nicaraguensis sp. n., L. opela Roback, L. panamensis sp. n., L. parabecki Roback, L. parafittkaui sp. n., L. parareniformis sp. n., L. paravirescens sp. n., L. paulae sp. n., L. pilosella Loew, L. reniformis sp. n., L. robacki sp. n., L. semicurvata sp. n., L. setosa sp. n., L. separata Edwards, L. sofiae sp. n., L. tenata Roback, L. trilucida sp. n., L. unicolor sp. n., L. virescens Beck et Beck. A diagnosis of the genus is presented and morphological differences to closely related genera are discussed.