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The non-biting midge subfamily Tanypodinae (Diptera: Chironomidae) is species-rich, ecologically diverse, and near-globally distributed. Within the subfamily, aspects of generic and species-level taxonomy remain poorly understood, in particular the validity of assignment of Australian and New Zealand taxa to genera erected for northern hemisphere (Holarctic) fauna. Here, we place the austral diversity within this global context by extensive geographical and taxonomic sampling in concert with a multilocus phylogenetic approach. We incorporated sequence data for mitochondrial COI, and nuclear 28S and CAD, and conducted Bayesian and maximum likelihood phylogenetic inferences and Bayesian divergence time estimation. The resolved phylogeny supported many associations of Australian taxa with their proposed Holarctic congeners, with the exception of Apsectrotanypus Fittkau, and validates several taxa as endemic. Three of four New Zealand sampled taxa had their sister groups in Australia; New Zealand Monopelopia Fittkau was sister to a German congener. This included the first record of Procladius Kieffer from New Zealand. Most nodes connecting austral and Holarctic taxa clustered around the Cretaceous-Tertiary boundary (60-80 mya), whereas New Zealand-Australia nodes were generally slightly younger (53-57 mya). Together, these data contribute substantially to our understanding of the taxonomy, systematics and biogeography of the Australian Tanypodinae and more broadly to knowledge of Australia's aquatic insect biodiversity., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The diversity and endemism of Australian Tanypodinae (Diptera: Chironomidae) has been unclear from morphological comparisons with well-grounded northern hemisphere taxonomy. As part of a comprehensive study, here we focus on one of the few described endemic genera, Yarrhpelopia Cranston. Extensive and intensive new sampling and newly-acquired molecular data provides clarity for the type species, Yarrhpelopia norrisi Cranston and allows recognition of congeners and potential sister group(s). We describe Yarrhpelopia acorona Cranston Krosch sp. n., and we recognise a third species from Western Australia, retaining an informal code 'V20' due to inadequate reared / associated material for formal description. We recognise a robust clade Coronapelopia Cranston Krosch gen. n., treated as a genus new to science for two new species, Coronapelopia valedon Cranston Krosch sp. n. and Coronapelopia quadridentata Cranston Krosch sp. n., from eastern Australia, each described in their larval and pupal stages and partial imaginal stages. Interleaved between the independent new Australian clades Yarrhpelopia and Coronapelopia are New World Pentaneura and relatives, that allow a tentative inference of a dated gondwanan (austral) connection. Expanded sampling indicates that Y. norrisi, although near predictably present in mine-polluted waters, is not obligate but generally indicates acidic waters, including natural swamps and Sphagnum bogs. The inferred acidophily, including in drainages of mine adits, applies to many taxa under consideration here.

The immature stages of the New Zealand endemic Paucispinigera Freeman and Paraborniella Freeman, endemic to Australia, are described fully for the first time. A new species of Paratendipes Kieffer endemic to Australia is described in all life stages. Morphological support from larvae for placement in a 'Microtendipes' group in tribe Chironomini includes the 6-segmented antenna with Lauterborn organs on the 2nd and 3rd segments, and a well-demarcated, multi-toothed ventromentum. The pupa has a few-branched thoracic horn and the distribution of taeniate L-setae is informative. Significant differentiating characters of the adult male include the form of tibial apices and the structures of superior and median volsellae in the genitalia. In the wing of Paucispinigera, the squama lacks setae, contrary to the original description, and unusually has vein M1+2 setose in both sexes and also the M3+4 setose in the female. An apical spine on the gonostylus of Paucispinigera is unusual. The female genitalia are described here for the first time but lack phylogenetically informative variation. An Australian species of the widespread genus Paratendipes Kieffer, described here as Paratendipes sinespina sp. n., is provided for comparison. Paucispinigera and Paraborniella each warrant independent generic rank.

Identification keys are provided for the final (4th) instar larvae of genera of Chironomidae (Diptera), from aquatic habitats in Australia and New Zealand. Morphological features of taxonomic utility are discussed and illustrated by line drawings. Summaries of described species for each genus and their distribution is provided, with reference to means of further identification where available. In the subfamily Podonominae, 5 genera are keyed of which 3 are recorded from New Zealand; the 4 genera of Aphroteniinae are from Australia (absent from New Zealand); in Diamesinae 1 genus is Australian, 2 are from New Zealand; in the Tanypodinae 21 genera are found in Australia and 4 are from New Zealand; in Orthocladiinae 31 genera are reported from Australia, 14 from New Zealand; and in Chironominae 43 genera are keyed from Australia, 9 from New Zealand. Larvae of Axarus Roback, Chernovskiia Sæther and Omisus Townes (Chironomini) are recognised in Australia for the first time. The undescribed larva of Paucispinigera Freeman, endemic to New Zealand, is keyed and several other New Zealand taxa are included based on unpublished records. Genera reported from Australia and New Zealand as adults, but unknown as larvae, are listed.

Riethia Kieffer, known previously from New Zealand for a species stated to be also in Australia plus several Australian and South American species, is revised for the Austro-Pacific region. The three previously-described Australian species Riethia stictoptera Kieffer (the genotype), Riethia cinctipes Freeman and Riethia plumosa Freeman are distinct and valid, and are redescribed in all stages. In contrast, Riethia zeylandica Freeman now is restricted to New Zealand: Australian specimens previously allocated to R. zeylandica belong to several new species recognised on morphology of adult male, pupa and larva, with guidance from molecular data. Most belong to a widespread eastern Australian Riethia azeylandica sp. n.: others are allocated to Riethia hamodivisa sp. n., Riethia paluma sp. n., Riethia phengari sp. n. and Riethia queenslandensis sp. n., each with a more restricted range. From Western Australia three species, Riethia donedwardi sp. n., Riethia noongar sp. n. and Riethia wazeylandica sp. n., are described as new from adult male, pupa and larva. Riethia kakadu sp. n. is described from the monsoonal tropics of Northern Territory from the adult male and tentatively associated pupa. From New Caledonia a reared species is described as Riethia neocaledonica sp. n.. Illustrated identification keys are provided for the males, pupae and larvae. Unassociated larvae that key to reared described species are excluded from type status, and based on morphology and molecular evidence three unreared larval types, 'A', 'B' and 'C', are also described and keyed. Previously reported molecular vouchers are reviewed, and certain Genbank accessions re-identified. Extensive data shows Riethia are distributed almost throughout Australia from standing and flowing waters, from tropics and subtropics to cool temperate Tasmania, but probably only in permanent and standing waters. The immature stages of several taxa can co-occur: as many as four can be found simultaneously in one site. Terminology of the volsellae of the male genitalia and the dorsal head and maxilla of the larva is reviewed.

Kribiodorum Kieffer, an otherwise North American and African genus of Chironomini (Diptera: Chironomidae), extends to the Oriental region through two new species. An adult male and female of Kribiodorum malicky sp. n. is newly described from Thailand, and from Brunei (Borneo) a pharate male and the pupa of Kribiodorum belalong sp. n. is described. Additionally, from Namibia (s.w. Africa) a 'manuscript' taxon is described formally with co-authorship of the late Arthur Harrison as Kribiodorum kunene sp. n. Males of the new species and the sole new pupa conform substantially to generic diagnoses based on the North American Kribiodorum perpulchrum (Mitchell). Examination of specimens of African Kribiodorum pulchrum Kieffer and N. American K. perpulchrum confirms their morphological similarity and reaffirms the junior synonymy of Stelechomyia Reiss designated for the North American species. Kribiodorum expands the number of genera of Chironomidae with African and Asian representatives, although unusual in its absence from Australia yet presence in the Nearctic.

Skusella Freeman, 1961 (Diptera: Chironomidae, Chironominae), known previously from adults from Australia and Africa, is revised with extended descriptions, including for immature life stages. Skusella is diagnosed based on its type species S. subvittata (Skuse, 1889) from Australia, S. pallidipes (Kieffer, 1921) from the Afrotropical region (the only other originally included species), a second African species S. freemani Harrison, 2002, and S. silingae Tang, sp. n., newly described here from the pupa and adult males from China. The immature stages of Skusella have been known informally for several decades, notably for a characteristic fringe of setae on one or more abdominal pleurae of the pupa, shared only with newly described Paraskusella Cranston, 2018 within the Chironominae. Amongst larvae of Chironomini with 6-segmented antenna, alternate Lauterborn organs and a well-demarcated ventromentum, those of Skusella are distinguishable only with caution due to insufficient reared associations. Unassociated pupal exuviae, tentatively belonging to three unknown new species, are described informally from China, as is a larval type from Africa. Range extensions include for S. freemani, with pupae (newly described here) from Nigeria and Cameroon, at least 5000 km from the type locality in South Africa. A wider distribution of S. subvittata in Australia and Asia is revealed by extensive pupal exuviae sampled from drift.

The male, female and pupa of Nilodosis austrosinensis Tang & Cranston, sp. n., reared from 2 reservoirs of south China, are described. The male can be separated from congeners by the presence of few squamal setae, a relatively long spur on the mid- and hind legs, a characteristic superior volsella and distinctive pattern of all legs. The female can be separated by features of genitalia, namely the absence of a ventrolateral lobe and the dorsomesal lobe with the apex usually curved. The pupa is separable by a bare tergite VIII and by characteristic spinulation of the sternites. The generic diagnosis is emended and some problems alluded to. This is the first formal record of Nilodosis from the Oriental region, indeed the first outside the Afrotropical region.

For over 25 years an undescribed Tanypodinae (Diptera: Chironomidae) has been known to dominate the lotic invertebrate assemblage associated with long-term polluting mine adits in Captains Flat, on the Molonglo River, southern New South Wales, Australia. Although known in all life stages, it has been impossible to allocate the species to any described genus. Renewed interest in the taxonomy of the Tanypodinae, particularly associated with molecular investigations and pollution indicator status warrants formal description. All stages conform to tribe Pentaneurini, but each life stage differs in morphological resemblance. Yarrhpelopia Cranston gen. n. is proposed for the taxon previously referred to under the informal code name of 'genus A'. The genus name derives from south-east Australian aboriginal word yarrh, in recognition of its core distribution and presence in flowing waters. A single species, A. norrisi Cranston sp. n., is described, acknowledging the late Professor Richard Norris, an influential Australian limnologist. Larvae dominate the benthos immediately adjacent to mine adits that continue to leach heavy metals (zinc, cadmium, copper and lead) into downstream sediments. A wider distribution includes cleaner near pristine, eastern Australian rivers between 30° and 42°S, but these records are excluded from the type series pending molecular insights into species limits.

The immature stages are described for the first time for Chironomus (Xenochironomus) australiensis Freeman (Diptera: Chironomidae) and the adult male is redescribed including from type specimens. The species does not belong to Chironomus Meigen or Xenochironomus Kieffer, but is best placed in a modestly expanded Einfeldia Kieffer. Application of this genus name is clarified, including by a lectotype fixation for its type species, E. pectoralis Kieffer, 1924. Einfeldia australiensis (Freeman) comb. n. provides the first record of the genus from Australia; otherwise the genus is reported confidently only from North America, Central America and western Europe to Japan. The immature stages of E. australiensis occur in relatively shallow mesotrophic to eutrophic dune lakes and maars with circum-neutral pH and high conductivity, from southeastern Queensland to southern Australia. The cytology is described briefly from larval salivary glands. Alternative genus placements for the species are discussed, and problems with Einfeldia and connected systematics in the tribe Chironomini are addressed.

The presence of the Afro-Australian genus Conochironomus Freeman, 1961 (Diptera: Chironomidae) in Asia has been recognised only informally. An unpublished thesis included Conochironomus from Singapore, and the genus has been keyed from Malaysia without named species. Here, the Sumatran Conochironomus tobaterdecimus (Kikuchi & Sasa, 1980) comb. n. is recorded from Singapore and Thailand. The species is transferred from Sumatendipes Kikuchi & Sasa, 1980, rendering the latter a junior synonym (syn. n.) of Conochironomus Freeman. Conochironomus nuengthai sp. n. and Conochironomus sawngthai sp. n. are described as new to science, based on adult males from Chiang Mai, Thailand. All species conform to existing generic diagnoses for all life stages, with features from male and female genitalia, pupal cephalic tubercles and posterolateral 'spurs' of tergite VIII providing evidence for species distinction. Some larvae are linked to C. tobaterdecimus through molecular barcoding. Variation in other larvae, which clearly belong to Conochironomus and are common throughout Thailand, means that they cannot be segregated to species. Larval habitats include pools in river beds, urban storage reservoirs, drains with moderately high nutrient loadings, and peat swamps. Endochironomus effusus Dutta, 1994 from north-eastern India may be a congener but may differ in adult morphology, thereby precluding formal new combination until discrepancies can be reconciled. Many problems with vouchering taxonomic and molecular material are identified that need to be rectified in the future.

Polypedilum nubifer (Skuse, 1889), originally described from Australia, is an apparently widespread species of Chironomidae (Diptera) that can attain nuisance densities in some eutrophic water bodies. Appropriate management depends upon the identity and ability to distinguish from potential cryptic taxa. A morphological study of larvae, pupae and adults of both sexes confirmed P. nubifer as widely distributed and frequently abundant, but also revealed two previously cryptic species of limited distribution in northern Australia. These species are described as new and illustrated in all stages here. Polypedilum quasinubifer Cranston sp. n. is described from north-west Queensland, Australia and also from Thailand and Singapore. Polypedilum paranubifer Cranston sp. n. is known only from retention ponds of a uranium mine in Northern Territory, Australia. Unusual morphological features of P. nubifer including alternate Lauterborn organs on the larval antenna, cephalic tubules on the pupa and frontal tubercles on the adult head are present in both new species as well. Newly slide-mounted types of Polypedilum pelostolum Kieffer, 1912 (lectotype designated here) confirm synonymy to Chironomus nubifer Skuse, 1889, examined also as newly-slide mounted types. Reviewed plus new evidence does not support recognition of Tripedilum Kieffer, 1921 as a separate taxon; therefore, Tripedilum is returned to junior synonymy with Polypedilum s. str.

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).

The Australian species of the Orthocladiinae genus Cricotopus Wulp (Diptera: Chironomidae) are revised for larval, pupal, adult male and female life stages. Eleven species, ten of which are new, are recognised and keyed, namely Cricotopus acornis Drayson & Cranston sp. nov., Cricotopus albitarsis Hergstrom sp. nov., Cricotopus annuliventris (Skuse), Cricotopus brevicornis Drayson & Cranston sp. nov., Cricotopus conicornis Drayson & Cranston sp. nov., Cricotopus hillmani Drayson & Cranston, sp. nov., Cricotopus howensis Cranston sp. nov., Cricotopus parbicinctus Hergstrom sp. nov., Cricotopus tasmania Drayson & Cranston sp. nov., Cricotopus varicornis Drayson & Cranston sp. nov. and Cricotopus wangi Cranston & Krosch sp. nov. Using data from this study, we consider the wider utility of morphological and molecular diagnostic tools in untangling species diversity in the Chironomidae. Morphological support for distinguishing Cricotopus from Paratrichocladius Santo-Abreu in larval and pupal stages appears lacking for Australian taxa and brief notes are provided concerning this matter.

Based on associated material collected from Macau and Guangxi, the pupae and larvae of Polypedilum (Pentapedilum) nodosum Johannsen and P. (Tripodura) masudai Tokunaga are described completely for the first time. Both species are newly recorded from China. Characters for distinguishing the immature stage of these species from other allied Polypedilum species are noted. The previously recorded Polypedilum (Pentapedilum) 'K1' of Cranston  (1996) is shown to be P. nodosum. Information is provided on distribution and ecological tolerances.

Macroinvertebrates that are collected in large numbers pose major problems in basic and applied biodiversity research: identification to species via morphology is often difficult, slow and/or expensive. DNA barcodes are an attractive alternative or complementary source of information. Unfortunately, obtaining DNA barcodes from specimens requires many steps and thus time and money. Here, we promote a short cut to DNA barcoding, that is, a nondestructive PCR method that skips DNA extraction ('direct PCR') and that can be used for a broad range of invertebrate taxa. We demonstrate how direct PCR can be optimized for the larvae and adults of nonbiting midges (Diptera: Chironomidae), a typical invertebrate group that is abundant, contains important bioindicator species, but is difficult to identify based on morphological features. After optimization, direct PCR yields high PCR success rates (>90%), preserves delicate morphological features (e.g. details of genitalia, and larval head capsules) while allowing for the recovery of genomic DNA. We also document that direct PCR can be successfully optimized for a wide range of other invertebrate taxa that need routine barcoding (flies: Culicidae, Drosophilidae, Dolichopodidae, Sepsidae; sea stars: Oreasteridae). Key for obtaining high PCR success rates is optimizing (i) tissue quantity, (ii) body part, (iii) primer pair and (iv) type of Taq polymerase. Unfortunately, not all invertebrates appear suitable because direct PCR has low success rates for other taxa that were tested (e.g. Coleoptera: Dytiscidae, Copepoda, Hymenoptera: Formicidae and Odonata). It appears that the technique is less successful for heavily sclerotized insects and/or those with many exocrine glands., (© 2014 John Wiley & Sons Ltd.)

Many insect clades, especially within the Diptera (true flies), have been considered classically 'Gondwanan', with an inference that distributions derive from vicariance of the southern continents. Assessing the role that vicariance has played in the evolution of austral taxa requires testing the location and tempo of diversification and speciation against the well-established predictions of fragmentation of the ancient super-continent. Several early (anecdotal) hypotheses that current austral distributions originate from the breakup of Gondwana derive from studies of taxa within the family Chironomidae (non-biting midges). With the advent of molecular phylogenetics and biogeographic analytical software, these studies have been revisited and expanded to test such conclusions better. Here we studied the midge genus Stictocladius Edwards, from the subfamily Orthocladiinae, which contains austral-distributed clades that match vicariance-based expectations. We resolve several issues of systematic relationships among morphological species and reveal cryptic diversity within many taxa. Time-calibrated phylogenetic relationships among taxa accorded partially with the predicted tempo from geology. For these apparently vagile insects, vicariance-dated patterns persist for South America and Australia. However, as often found, divergence time estimates for New Zealand at c. 50 mya post-date separation of Zealandia from Antarctica and the remainder of Gondwana, but predate the proposed Oligocene 'drowning' of these islands. We detail other such 'anomalous' dates and suggest a single common explanation rather than stochastic processes. This could involve synchronous establishment following recovery from 'drowning' and/or deleteriously warming associated with the mid-Eocene climatic optimum (hence 'waving', which refers to cycles of drowning events) plus new availability of topography providing of cool running waters, or all these factors in combination. Alternatively a vicariance explanation remains available, given the uncertain duration of connectivity of Zealandia to Australia-Antarctic-South America via the Lord Howe and Norfolk ridges into the Eocene., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Donald Henry Colless (24 August 1922–16 February 2012) was a taxonomist at the Australian National Insect Collection (ANIC) from 1960 until his retirement in 1987. He continued working in ANIC as an Honorary Fellow until his death in 2012. Don’s main scientific interests were in the taxonomy and biology of true flies, and in the theory of phylogenetic reconstruction and classification. Don was trained in entomology at the University of Sydney, and spent nearly two decades of his early career in Asia studying mosquitoes and disease transmission, first in the Army during the Second World War in New Guinea and Borneo (1942–45), then after the war in North Borneo (1947–1952) and as a lecturer in the Department of Parasitology at the University of Malaya (1952–1960) in Singapore. We list the 127 scientific papers and book chapters that Don published during his scientific career that spanned 64 years. Six of these papers were published in the prestigious international journal Nature, and he was Chief Curator of the ANIC from 1971–1977. Don had extremely broad taxonomic interests, publishing on the taxonomy of 18 families of Diptera that spanned the phylogenetic breadth of the order. He described as new to science the fly families Perissommatidae and Axiniidae, thirteen new genera and over 120 species and, with David McAlpine, authored the Diptera chapters in both editions of The Insects of Australia (Melbourne University Press, 1970 and 1991). He published a number of influential critiques of cladistic theory in the 1960's and 1970's, and advocated a phenetic approach to the discovery of taxonomic groups, and phylogenetic reconstruction.

Barbadocladius n. gen. is erected and described in larval, pupal and adult stages for two species: B. andinus sp. nov. and B. limay sp. nov., from Andean streams. The larva is distinctive by virtue of the very large ventromental 'beard' and the anterior parapods with a 'sleeve' of hooklets in addition to apical pectinate claws. The pupa has hooklets on some tergal and sternal intersegmental membranes. The adult, reported only in teneral specimens has hairy eyes, no antennal apical strong seta, no acrostichals, bare and unmarked wings, cylindrical 4th tarsomere subequal in length to the 5th, pulvilli about half the claw length, and hypopygium with anal point, lacking a virga. Molecular phylogenetic analysis eliminates relationships directly to the Eukiefferiella complex (which also have pupal hooklets), or to the Cricotopus group (adults also with hairy eyes), suggesting instead a sister group relationship to a suite of predominantly austral genera of Orthocladiinae.

Restrictions to effective dispersal and gene flow caused by the fragmentation of ancient supercontinents are considered to have driven diversification and speciation on disjunct landmasses globally. Investigating the role that these processes have played in the development of diversity within and among taxa is crucial to understanding the origins and evolution of regional biotas. Within the chironomid (non-biting midge) subfamily Orthocladiinae (Diptera: Chironomidae), a group of genera that are distributed across the austral continents (Australia, New Zealand, South America) have been proposed to represent a relict Gondwanan clade. We used a molecular approach to resolve relationships among taxa with the aim to determine the relative roles that vicariance and dispersal may have played in the evolution of this group. Continental biotas did not form monophyletic groups, in accordance with expectations given existing morphological evidence. Patterns of phylogenetic relationships among taxa did not accord with expected patterns based on the geological sequence of break-up of the Gondwanan supercontinent. Likewise, divergence time estimates, particularly for New Zealand taxa, largely post-dated continental fragmentation and implied instead that several transoceanic dispersal events may have occurred post-vicariance. Passive dispersal of gravid female chironomid adults is the most likely mechanism for transoceanic movement, potentially facilitated by West Wind Drift or anti-cyclone fronts. Estimated timings of divergence among Australian and South American Botryocladius, on the other hand, were congruent with the proposed ages of separation of the two continents from Antarctica. Taken together, these data suggest that a complex relationship between both vicariance and dispersal may explain the evolution of this group. The sampling regime we implemented here was the most intensive yet performed for austral members of the Orthocladiinae and unsurprisingly revealed several novel taxa that will require formal description., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Australasia, which consists of Australia and the adjacent islands of the southwestern Pacific Ocean, has an insect diversity approximately proportional to the land mass. This diversity is distinctive, with some major groups missing and others having radiated. Iconic species are familiar to most people living in Australia and New Zealand, and a range of insects once contributed to Aboriginal Australian culture and diet. Conservation of Australasian entomological biodiversity is an increasing challenge for contemporary scientists. Examples are provided of insect conservation schemes from New Guinea, New Zealand, and Australia. Funding for insect biodiversity studies beyond flagship species is needed.

Biological monitoring requires identification of the fauna of the environment under study. Three prevalent methods have been used to alleviate the need for universal species-level identification: selection of indicator taxa, taxonomic reduction, and allocation to functional groups. Each method is discussed in relation to species recognition. It is suggested that the validity of all attempts to reduce data should be tested in the light of phylogenetic hypotheses.

Hypersensitivity to Chironomidae (non-biting midges) has been a problem in the Sudan since about 1927 and appears to be due to increased breeding of a single chironomid species, Cladotanytarsus lewisi (Freeman). Mass emergence of the midges in thought to be related to the larval diet of algae and diatoms, the numbers of which are greatly enhanced by the retention of plant nutrients in lacustrine conditions resulting from interruption to the natural flow of the Nile by the construction of dams. Immunological studies in allergic individuals using an allergen extract prepared from C. lewisi indicate that the concentration of specific immunoglobulin E ("allergic antibody") directed against C. lewisi is raised in patients with established hypersensitivity to the midge but not in control subjects. The concentration of specific IgE is also related to the severity of clinical symptoms. These results indicate that this widespread and important "man made" hypersensitivity in the Sudan has the features of well recognized immediate-type allergy commonly associated with pollens and other air-borne allergens.

Chironomids (non-biting midges) are known to cause IgE-mediated hypersensitivity in man. This study compares the cross-reactivity between the chironomid midge Cladotanytarsus lewisi ('green nimitti'), a widespread cause of allergy in the Sudan and Chironomus riparius (= thummi, CTT) where larvae are used as pet fish food and where haemoglobins were previously shown to be major allergens. As with C. riparius, immature forms of C. lewisi also contain allergenic material since skin test responses to larval, pupal and adult extracts were obtained in Sudanese individuals. Crossed radioimmunoelectrophoresis of the C. lewisi larval and pupal extracts indicate that they contain a higher proportion of the allergenic fractions than adults. Further evidence of common allergen determinants between C. lewisi and C. riparius were obtained by the demonstration of positive skin-prick tests, in Sudanese patients, to extracts of larval, adult and isolated haemoglobin extracts of C. riparius. Cross-reactivity between C. lewisi and C. riparius was also demonstrable by RAST inhibition studies. A dose-dependent inhibition was observed using both the C. lewisi adult midge RAST and the C. riparius haemoglobin RAST, the two respective antigens, and sera from individuals hypersensitive to either C. lewisi or C. riparius. Due to the immunological cross-reactivity found between these distantly related species, we conclude that chironomids should be seen as significant environmental and occupational allergens.

Skin 'prick' tests with an extract of the 'green nimitti' midge, Cladotanytarsus lewisi (Freeman), a potent airborne allergen associated with asthma and rhinitis in the Khartoum area, were undertaken on asthmatic subjects in several localities in the Sudan and Egypt in order to provide further information on the extent of this allergic problem. In the Sudan skin-test-positive individuals were identified in the Khartoum area, Kosti, Sennar, Wad Medani, Shendi and Atbara, and in Egypt at Aswan, Luxor and Qena. A number of asthmatic individuals giving a positive reaction to C. lewisi were also tested with extracts of seven sub-dominant species of Nilotic Chironomidae. There appeared to be a limited degree of cross-reactivity with these species, particularly Dicrotendipes fusconotatus (Kieffer), Procladius noctivagus (Kieffer) and Conchapelopia cygnus (Kieffer). The numbers of C. lewisi sensitive subjects giving positive skin-tests to these other species were ten out of 20, 12 out of 28 and 12 out of 29 respectively. In contrast, only five out of 31 reacted to Paracladopelma graminicolor (Kieffer), five out of 24 to Cladotanytarsus pseudomancus (Goetghebuer), three out of 32 to Nanocladius vitellinus (Kieffer) and two out of 28 to Cryptochironomus neonilicola (Freeman). These results indicate that Cladotanytarsus lewisi is a major source of chironomid allergen(s) in these asthmatic subjects and that hypersensitivity to C. lewisi is probably far more widespread than originally envisaged.

Some chironomid (non-biting) midges contain potent human allergens, principally in the form of insect haemoglobins. In view of previously demonstrated immunological cross-reactivity between some different midge species, the possible role of British midges in allergic disease was studied. The chosen site, Farmoor near Oxford, is a village that has been subject to several annual nuisance midge swarms since the construction of an adjacent water-supply reservoir in 1964, which was extended in 1975. The incidence and severity of hypersensitivity in the community was assessed by questionnaire survey, skin-prick testing, radio-allergosorbent tests (RAST) and RAST inhibition, and enzyme-linked immunosorbent assays (ELISA) and ELISA inhibition, using extracts of locally caught adult midges (Tanytarsus sylvaticus, T. bathophilus and Chironomus anthracinus) and laboratory cultured larvae (C. riparius). These tests revealed a low but significant level of midge-related hypersensitivity with skin-prick results showing seasonal fluctuations, but this seasonality was not confirmed with an IgE RAST. A correlation between IgE levels and relevant symptoms was found (P less than 0.01) using RAST, but not between IgE and skin-test results. Enzyme-linked immunosorbent assay results did not correlate with any other parameter measured. The incidence and severity of allergic reactions to the midges tested in this community was low.

Allergens in extracts of the 'green nimitti' midge, Cladotanytarsus lewisi Freeman (Diptera: Chironomidae), a cause of widespread hypersensitivity in the Sudan, were isolated and partially characterized by assays which depend on the binding of 125I-anti-IgE to allergen-IgE complexes. These methods included RAST inhibition, crossed radioimmunoelectrophoresis (CRIE) and rocket radioimmunoelectrophoresis (RRIE). Following Sephadex G100 chromatography the 'major peak' of allergenicity as determined by RAST inhibition, RRIE and SDS-PAGE was associated with molecules of approximately 17 000 daltons. The peak eluting at Vo contained material of molecular weight 66 000 daltons which also bound 125I-anti-IgE, but had only 61% of the activity of the 'major peak' by RAST inhibition. By isoelectric focusing and RRIE of fractions obtained by chromatofocusing with polybuffer exchanger 94, the 'major peak' was associated with multiple bands with a pI range of 3.5-5.5. These results indicate that the major allergens from C. lewisi are a group of closely related acidic peptides.

Chironomid hemoglobins are potent allergens. The allergenic and antigenic activities of these hemoglobins are studied with the help of RAST, RAST inhibition and double immunodiffusion. Human as well as rabbit antisera were used. It was shown that hemoglobins are the main antigenic/allergenic components in extracts of Camptochironomus tentans larvae. Furthermore, immunological cross-reactivity among larvae, pupae and adult midges of this species are shown to be due to the existence of hemoglobin antigenic determinants in all developmental stages of this insect.

Chironomid midges are small (2-15 mm) non-biting flies, characteristically seen swarming by water at dusk. Allergens of the "green nimitti" midge, Cladotanytarsus lewisi (Freeman) (Diptera: Chironomidae), a cause of widespread hypersensitivity in the Sudan, were isolated and partially characterized by Sephacryl S200 chromatography. The allergenicity of the fractions was identified by "rocket" autoradiography, RAST inhibition, skin "prick" tests, and the immunoblot technique. The fractions were further analysed by isoelectric focusing and SDS-PAGE. Two major allergens with pI's ranging from 4.3 to 6.0 were identified and had molecular weights of approximately 17,000 and 32,000 daltons, sizes compatible with their being monomeric and dimeric haemoglobins. Since chironomids occur in nuisance numbers worldwide and their haemoglobins have been shown to produce severe hypersensitivity reactions in man, they should be seen as an important potential cause of environmental and occupational allergy.

Various genera of non-biting midges (Diptera: Chironomidae) possess haemoglobins in larvae and adults. For certain species, these haemoglobins have been implicated in human allergic disease in several countries. The present study confirms and extends observations that haemoglobin is present in many species of Chironomidae, establishes that it is retained from the larval to the adult stage and shows that haemoglobin persists both in live and in dead dry flies. Previous suggestions that Chironomidae should be seen as important environmental and occupational allergens are clearly substantiated.