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Blue shining fungus gnats (Diptera) had been long reported in the Waitomo caves of New Zealand (Arachnocampa luminosa Skuse), in stream banks of the American Appalachian Mountains (Orfelia fultoni Fisher) in 1939 and in true spore eating Eurasiatic Keroplatus Bosc species. This current report observes that similar blue light emitting gnat larvae also occur nearby the Betary river in the buffer zone of High Ribeira River State Park (PETAR) in the Atlantic Forest of Brazil, where the larvae were found when on fallen branches or trunks enveloped in their own secreted silk. The new species is named Neoceroplatus betaryiensis nov. sp. (Diptera: Keroplatidae: Keroplatinae: Keroplatini) based on a morphological analysis. Neoceroplatus betaryiensis nov. sp. larvae emit blue bioluminescence that can be seen from their last abdominal segment and from two photophores located laterally on the first thoracic segment. When touched, the larvae can actively stop its luminescence, which returns when it is no longer being agitated. The in vitro bioluminescence spectrum of N. betaryiensis nov. sp. peaks at 472 nm, and cross-reactivity of hot and cold extracts with the luciferin-luciferase from Orfelia fultoni indicate significant similarity in both enzyme and substrate of the two species, and that the bioluminescence system in the subfamily Keroplatinae is conserved.

Bioluminescence in Diptera is found in the Keroplatidae family, within Arachnocampininae and Keroplatinae subfamilies, with reported occurrences in Oceania, Eurasia, and Americas. Larvae of Orfelia fultoni, which inhabit stream banks in the Appalachian Mountains, emit the bluest bioluminescence among insects, using it for prey attraction, similarly to Arachnocampa spp. Although bioluminescence has a similar prey attraction function, the systems of Arachonocampininae and Keroplatinae subfamilies are morphologically/biochemically distinct, indicating different evolutionary origins. To identify the possible coding genes associated with physiological control, ecological adaptations, and origin/evolution of bioluminescence in the Keroplatinae subfamily, we performed the RNA-Seq analysis of O. fultoni larvae during day and night and compared it with the transcriptomes of Arachnocampa luminosa, and reanalyzed the previously published proteomic data of O. fultoni against the RNA-Seq dataset. The abundance of chaperones/heat-shock and hexamerin gene products at night and in luciferase enriched fractions supports their possible association and participation in bioluminescence. The low diversity of copies/families of opsins indicate a simpler visual system in O. fultoni. Noteworthy, gene products associated with silk protein biosynthesis in Orfelia were more similar to Lepidoptera than to the Arachnocampa, indicating that, similarly to the bioluminescent systems, at some point, the biochemical apparatus for web construction may have evolved independently in Orfelia and Arachnocampa.

Animals use adhesive secretions in highly diverse ways, such as for settlement, egg anchorage, mating, active or passive defence, etc. One of the most interesting functions is the use of bioadhesives to capture prey, as the bonding has to be performed within milliseconds and often under unfavourable conditions. While much is understood about the adhesive and biomechanical properties of the threads of other hunters such as spiders, barely anything is documented about those of the New Zealand glowworm Arachnocampa luminosa. We analysed tensile properties of the fishing lines of the New Zealand glowworm Arachnocampa luminosa under natural and dry conditions and measured their adhesion energy to different surfaces. The capture system of A. luminosa is highly adapted to the prevailing conditions (13–15 °C, relative humidity of 98%) whereby the wet fishing lines only show a bonding ability at high relative humidity (>80%) with a mean adhesive energy from 20–45 N/m and a stronger adhesion to polar surfaces. Wet threads show a slightly higher breaking strain value than dried threads, whereas the tensile strength of wet threads was much lower. The analyses show that breaking stress and strain values in Arachnocampa luminosa were very low in comparison to related Arachnocampa species and spider silk threads but exhibit much higher adhesion energy values. While the mechanical differences between the threads of various Arachnocampa species might be consequence of the different sampling and handling of the threads prior to the tests, differences to spiders could be explained by habitat differences and differences in the material ultrastructure. Orb web spiders produce viscid silk consisting of β-pleated sheets, whereas Arachnocampa has cross-β–sheet crystallites within its silk. As a functional explanation, the low tear strength for A. luminosa comprises a safety mechanism and ensures the entire nest is not pulled down by prey which is too heavy.

Bioluminescence in Diptera is found in the family Keroplatidae, in the glowworms of the genera Arachnocampa, Orfelia and Keroplatus. Despite belonging to the same family, Arachnocampa spp. and Orfelia fultoni display morphologically and biochemically distinct bioluminescence systems: Arachnocampa spp. produce light by the terminal ends of Malpighian tubules using ATP, a luciferin and a luciferase, whereas Orfelia fultoni produces light by translucent areas associated with rows of black bodies in the anterior and posterior parts of the body, using a 140 kDa luciferase and a luciferin which do not cross-react with the Arachnocampa luciferin–luciferase system, and a substrate binding fraction (SBF) which apparently releases luciferin in the presence of reductants. While several other keroplatids are not luminescent, we recently discovered a non-luminescent web-constructing keroplatid larva living in the roofs of caves in the Atlantic rainforest in Brazil, which noteworthily has a compound with Orfelia luciferin-like activity and its associated binding protein (SBF). Both the Neoditomyia luciferin-like compound and SBF cross-react with purified Orfelia luciferase to produce light in the same blue region of the Orfelia luciferin–luciferase system (479 nm). We also checked for the presence of Orfelia-type luciferin in Arachnocampa luminosa and Aedes aegytpi larval bodies, but no traces were found. Molecular studies indicate that Neoditomyia sp. is phylogenetically closer to Keroplatus and Orfelia than to Arachnocampa species. The presence of luciferin and its associated binding protein in this non-bioluminescent keroplatid larva indicates that luciferin may display another important biochemical function in keroplatid larvae and suggests that bioluminescence could be a recently evolved trait in Keroplatidae.

The New Zealand glowworm, Arachnocampa luminosa, is well-known for displays of blue-green bioluminescence, but details of its bioluminescent chemistry have been elusive. The glowworm is evolutionarily distant from other bioluminescent creatures studied in detail, including the firefly. We have isolated and characterised the molecular components of the glowworm luciferase-luciferin system using chromatography, mass spectrometry and 1H NMR spectroscopy. The purified luciferase enzyme is in the same protein family as firefly luciferase (31% sequence identity). However, the luciferin substrate of this enzyme is produced from xanthurenic acid and tyrosine, and is entirely different to that of the firefly and known luciferins of other glowing creatures. A candidate luciferin structure is proposed, which needs to be confirmed by chemical synthesis and bioluminescence assays. These findings show that luciferases can evolve independently from the same family of enzymes to produce light using structurally different luciferins.

Fungus-gnats of the genus Arachnocampa are unique among bioluminescent insects for displaying blue–green bioluminescence, and are responsible for one of the most beautiful bioluminescence spectacles on the roofs of the Waitomo Caves. Despite morphological studies showing that Arachnocampa larval lanterns involve specialization of the Malpighian tubules, the biochemical origin of their bioluminescence remains enigmatic. Using a cDNA library previously constructed from lanterns of the New Zealand glowworm A. luminosa, we carried out the first transcriptional analysis of ~ 500 expressed sequence tags (ESTs) to identify putative candidate proteins for light production, and to better understand the molecular physiology of the lanterns and their relationship with Malpighian tubule physiology. The analysis showed an abundance of hexamerin-like proteins, as well as luciferase-like enzymes, indicating a possible critical role for these proteins in bioluminescence. These findings were corroborated by proteomic analysis of lantern extracts, which showed the presence of hexamerins and luciferase-like enzymes. Other gene products typical of Malpighian tubules, such as detoxifying enzymes, were also found. The results support the existence of an evolutionary link between Malpighian tubule detoxification and the origin of bioluminescence in these Diptera. Copyright © 2015 John Wiley & Sons, Ltd.

Larvae of the weakly blue-luminescent fungus gnat Keroplatus nipponicus possess on either side of their heads a small black stemmatal eye with a plano-convex lens approximately 25 μm in diameter. In total, 12-14 retinula cells give rise to a centrally fused rhabdom of up to 8 μm in diameter. The rhabdom's constituent microvilli, approximately 70 nm in width, are roughly orthogonally oriented, a requirement for polarization sensitivity. Screening pigment granules are abundant in the retinula cells and measure at least 1 μm in diameter. In comparison with the stemmatal eye of the brightly luminescent Arachnocampa luminosa, that of K. nipponicus is considerably smaller with a poorer developed lens and a rhabdom that is less voluminous, but possesses wider microvilli. Although the larval eye of K. nipponicus can be expected to be functional, as the larvae react to light with a behavioural response, the eyes are probably mainly involved in the detection of ambient light levels and not, as in A. luminosa, also in responding to the luminescence of nearby conspecifics.

The behaviour of pupae and adults of the New Zealand glowworm Arachnocampa luminosa was observed in Reserve Cave and in the cave entrance at Waitomo, New Zealand, from remote video recordings made with infra-red light. One female pupa observed in the cave was videotaped for 111 h before the adult emerged and copulated with a male. This female took 13 h 23 min to eclose, during which she glowed intermittently on 17 occasions. The male alighted upon the emerging female 13 h and 8 min after eclosion commenced and 3 min 33 s after she last glowed. Copulation commenced 12 h 55 min later and continued for 12 h 36 min. Two minutes after the pair separated, both adults were caught and eaten by a predatory harvestman (Megalopsalis tumida). Most of the eclosion was recorded for a second female pupa observed, on a rock face in the bush-clad entrance to Reserve Cave after 97 h of videotaping. Prior to eclosion this pupa made flicking movements when small crawling invertebrates contacted it during the daytime; this is...

Background The New Zealand glowworm is the larva of a carnivorous fungus gnat that produces bioluminescence to attract prey. The bioluminescent system of the glowworm is evolutionarily distinct from other well-characterised systems, especially that of the fireflies, and the molecules involved have not yet been identified. We have used high throughput sequencing technology to produce a transcriptome for the glowworm and identify transcripts encoding proteins that are likely to be involved in glowworm bioluminescence. Results Here we report the sequencing and annotation of the first transcriptome of the glowworm, and a differential analysis of expression from the glowworm light organ compared with non-light organ tissue. The analysis identified six transcripts encoding proteins that are potentially involved in glowworm bioluminescence. Three of these proteins are members of the ANL superfamily of adenylating enzymes, with similar amino acid sequences to that of the luciferase enzyme found in fireflies (31 to 37 % identical), and are candidate luciferases for the glowworm bioluminescent system. The remaining three transcripts encode putative aminoacylase, phosphatidylethanolamine-binding and glutathione S-transferase proteins. Conclusions This research provides a basis for further biochemical studies into how the glowworm produces light, and a source of genetic information to aid future ecological and evolutionary studies of the glowworm. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2006-2) contains supplementary material, which is available to authorized users.

Two new species of harvestman (Opiliones: Neopilionidae: Enantiobuninae) are described from the Waitomo region of the North Island, New Zealand, Forsteropsalis bona sp. n. and F. photophaga sp. n. Both have been collected within caves in the region, where predation on glow-worms Arachnocampa luminosa has been previously recorded for one or both species (misidentified as ‘Megalopsalis tumida’). However, both are regarded as troglophiles rather than strict troglobites due to the presence of specimens outside the cave systems, and the absence of troglobitic adaptations. Megalopsalis tumida (Forster, 1944) is identified as a junior synonym of Forsteropsalis fabulosa (Phillipps & Grimmett, 1932).

The hypothesis that bioluminescence produced by larvae of the New Zealand glow-worm, Arachnocampa luminosa, attracts prey was tested experimentally in Reserve Cave, Waitomo, New Zealand, and in its bush-clad entrance over a total of 200 days during winter, spring, and summer. We compared catches on transparent adhesive traps placed either over glowworms or over areas from which glowworms had been removed. Adhesive traps over glowworms caught significantly more invertebrates per trap per day than did control traps. Glowworms in bush attracted greater numbers and types of invertebrates than did glowworms in the cave. Diptera predominated in both bush (86% of the total catch) and cave (89%). Also caught were small numbers of Araneae, Coleoptera, Hymenoptera, Orthoptera, Trichoptera, Gastropoda, Acarina, and Neuroptera—listed in order of abundance—but no adults of A. luminosa were caught. Glowworms under adhesive traps survived with little or no food for up to 78 days.

The temperature and relative humidity inside the Glowworm Cave are spatially and temporally variable to a degree atypical of other caves in temperate latitudes. Comparison between the climatic data for 1977-80 and for 1955 shows that the climate of the cave was much more stable in 1955 than it is at present. This paper suggests that the increase in climatic variability is closely related to the fact that since 1975, when the Upper Entrance was unblocked, the cave has behaved as a wind tunnel: the difference in density between the atmosphere inside and outside the cave allows the outside air to flow freely through the interior between the Upper and Lower Entrances. Climatic fluctuations are considered to be the main factor responsible for the decline in the total population of Arachnacampa tuminasa (Skuse) observed between 1975 and 1980. Climatic changes may also explain why the numbers and diversity of other cave fauna are low. A. tuminasa has five larval instars. Larval development is homodynami...

The swollen distal tips of the Malpighian tubules of the glow-worm Arachnocampa luminosa constitute the light organ. The ventral and lateral surfaces are covered by a tracheal ‘reflector’ and the nervous supply to the light organ comes from the ganglion in the penultimate segment. Fine nerve terminals, axons, and glial cells can be seen in close proximity to the basal surface of the cells of the light organ. The epithelial cells of the light organ are large, the cytoplasm dense, homogeneous and acidophilic. The cytoplasm gives a strong positive reaction for protein. The cytoplasm contains a high density of free ribosomes, patches of dense material, smooth endoplasmic reticulum, glycogen and scattered microtubules. Mitochondria are numerous; they are large, randomly distributed and packed with fine cristae. These cells lack the features characteristic of Malpighian tubule epithelial cells; infolding of the apical and basal cell surfaces is reduced and the cytoplasm contains few organelles. These cells do not contain secretory or photocyte granules and the grainy cell matrix is thought to be the luciferin substrate. Oxygen is supplied via the tracheal layer (which may have secondary reflecting properties) and light production controlled by neurosecretory excitation either directly via synapses, or by hormones. There are no other reports of Malpighian tubules of insects producing light and the fine structure of these cells is distinct. Thus, the swollen distal tips of the Malpighian tubules of the glow-worm undoubtedly constitute a unique luminescent organ.

Two entomopathogenic species of the hyphomycete genus Tolypocladium are described in detail. Tolypocladium extinguens sp. nov. was found on larvae of Arachnocampa luminosa (Diptera; Mycetophilidae) in caves in New Zealand. Tolypocladium cylindrosporum W. Gams is shown to be pathogenic to Aedes sierrensis and A. australis and is reported from Plecia nearctica (Diptera; Bibionidae). The occurrence of entomopathogenic species in this normally soil-borne genus is discussed.

The Malpighian tubules of the glow-worm are divided into four morphologically distinct regions, each composed of a different cell type. Part 3 of the Malpighian tubules of A. luminosa is intimately bound to the rectum by a layer of fat body. This association of the tubules with the hindgut is referred to as a cryptonephric system. This type of arrangement has been described in some Coleoptera and the larvae of most Lepidoptera but has never before been reported in the Diptera. In the glow-worm the cryptonephric tubules themselves are small, and adjacent to the fat body the epithelial cells are modified to form very thin windows or ‘leptophragmata’ (Lison, 1937). The main epithelial cells exhibit features characteristic of highly active, secretory Malpighian tubule cells. The high density of mitochondria and their association with all the microvilli is indicative of a highly active secretory cell. The high concentration of glycogen in these cells and their intimate association with the hindgut suggest that they may, in addition, have a nutrient absorptive function. The role of the cryptonephric rectal complex in the glow-worm is discussed in the light of present knowledge gained from previous studies of coleopteran and larval lepidopteran cryptonephric systems. On structural grounds a model is proposed for the regulation of the ionic environment of the rectum, and the uptake and metabolism of organic material from the rectal lumen by this cryptonephric complex.

The literature on Arachnocampa luminosa is reviewed together with a description of its taxonomic status. Information on other cave-dwelling keroplatids and mycetophilidsis also included.

The troglobitic harvestmenMegalopsalis tumidaandHendea myersi cavernicolainhabit the Waitomo Caves in New Zealand with their luminescent prey, the glow-wormArachnocampa luminosa. A distribution map of the harvestmen in the caves is presented. Both species of harvestman possess two prominent eyes on the cephalothorax, with lens diameters ofca. 500 μm and 250 μm forMegalopsalisandHendea, respectively. The eyes are of the everted (direct) type, with axons leaving the retina peripherally in a single optic nerve. Retinal organization is typical for harvestmen: rhabdomal groups are made up of three or four photoreceptive cells. Rhabdoms inMegalopsalisareca. 250 μm long and possess diameters of up to 20 μm, whereas the corresponding figures forHendeaare 150 μm and 26 μm. Movement of screening pigment granules into dark or light-adapted positions could be induced at any time of day.f-Numbers of 1.06 forMegalopsalisand 0.97 forHendealenses indicate considerable light-gathering power of the dioptric system, which does not appear to be wasted because of the massively developed, voluminous rhabdoms in the retina of both species. Electrophysiological recordings showed that both species are sensitive to a range of light intensities covering at least 5 log units of magnitude. In terms of electroretinogram (ERG)-determined spectral sensitivityMegalopsalisdisplayed high ultraviolet sensitivity and a secondary broad peak ranging from blue to green light, whereasHendeapossessed a clear green peak and secondary sensitivity to blue and ultraviolet radiation. Both species demonstrated an overall negatively phototactic response to a bright ultraviolet light source and a positively phototactic response to a dim, artificial ‘glow-worm’ light. Only three individuals ofMegalopsalisand noHendeawere caught in six automatic traps a few metres outside the cave entrance over a period of five months, but within the cave 92Megalopsalisand 174Hendeawere sampled in 12 collecting trips ofca. 4 h each, spread over one year. The relative lack of photoreceptor regression, despite the cave existence of the two species and their inability to produce light, is interpreted as a consequence of the light produced by the cave populations of glowworms.

It was established by means of the electroretinogramme method that eyes of male Arachnocampa luminosa respond to the presentation of monochromatic isophotonic light flashes with three response peaks. The eyes are maximally sensitive to light of 540 nm wavelength (green); a distinct second response peak exists to light of 460 nm wavelength (bluegreen); a third response peak exists in the near-ultraviolet. Light of a wavelength longer than 660 nm does not elicit measurable responses. The results are discussed in relation to eye anatomy and physiology as well as glowworm ecology and behaviour. It is concluded that the bluegreen light emitted by glowworm larvae is aimed at small aquatic prey, but that in the adult the light, “inherited” from the larval stage, obtained a new purpose — that of in-traspecific communication.

SYNOPSIS The New Zealand Glow-worm (Arachnocampa luminosa) is able suddenly to douse its light when alarmed. A suggestion is made as to how this could take place.

Both larval and adult New Zealand cave glowworms exhibit reactions to light; their photoreceptors must, therefore, be regarded as functional. The two principal stemmata of the larva possess large biconvex lenses and voluminous rhabdoms. Approximately 12 retinula cells are present. In light-adapted larvae the diameter of the rhabdom is 8 μm and that of an individual microvillus is 49.5 nm. Dark-adapted eyes have rhabdoms that measure 14 μm in cross section and microvilli with an average diameter of 54 nm. The compound eye of the adult comprises approximately 750 ommatidia, each with a facet diameter of 27–28 μm. A facet is surrounded by 1–6 interommatidial hairs which are up to 30 μm long. The interommatidial angle is 5.5°. Cones, consisting of 4 crystalline cone cells, are of the ‘acone’ type. Pigment granules in the primary pigment cells are twice as large as those of the retinula cells which measure 0.6–0.75 μm in diameter. The rhabdom is basically of the dipteran type, i.e. six open peripheral rhabdomeres surround 2 central rhabdomers arranged in a tandem position. The microvilli of cells 1–6 and cell 8 have diameters ranging from 68 to 73 nm, but those of the distally-located central rhabdomere 7 are 20% larger. This is irrespective of whether the eye is dark or light-adapted. In the latter the cones are long and narrow, the screening pigment granules closely surround the rhabdomeres, and the rhabdom is less voluminous than that of the dark-adapted eye.

Isoenzyme analysis of 11 isolates of Tolypocladium cylindrosporum and T. extinguens revealed considerable variability between strains of the same species and confirmed great differences between the two species. An exception was the striking similarities in isoenzyme patterns observed for the California isolates of T. cylindrosporum isolated from Aedes sierrensis larvae from 1972 to 1979. This considerable homogeneity suggests a rather stable genotypic equilibrium in the fungal population which was maintained with little alteration even after repeated subculturing in the laboratory.

Animals use adhesive secretions in a plethora of ways, either for attachment, egg anchorage, mating or as either active or passive defence. The most interesting function, however, is the use of adhesive threads to capture prey, as the bonding must be performed within milliseconds and under unsuitable conditions (movement of prey, variable environmental conditions, unfavourable attack angle, etc.) to be nonetheless successful. In the following study a detailed characterization of the prey capture system of the world-renowned glowworm group Arachnocampa from the macroscopic to the ultrastructural level is performed. The data reveal that the adhesive droplets consist mostly of water and display hygroscopic properties at varying humidity levels. The droplet core of Arachnocampa luminosa includes a certain amount of the elements sodium, sulphur and potassium (beside carbon, oxygen and nitrogen), while a different element composition is found in the two related species A. richardsae and A. tasmaniensis. Evidence for lipids, carbohydrates and proteins was negative on the histochemical level, however X-ray photoelectron spectroscopy confirm the presence of peptides within the droplet content. Different to earlier assumptions, the present study indicates that rather than oxalic acid, urea or uric acid are present in the adhesive droplets, presumably originating from the gut. Comparing the capture system in Arachnocampa with those of orb-spiders, large differences appear not only regarding the silky threads, but also, in the composition, hygroscopic properties and size of the mucous droplets.