Description of a New Species of Forcipomyia (Forcipomyia) (Diptera: Ceratopogonidae) and a Key to Species of the Subgenus From the Chinese Mainland.

Forcipomyia (Forcipomyia) makanensis Hou sp. n. (Diptera: Ceratopogonidae) is described and illustrated based on male and female specimens from China. It is characterized by the male aedeagus triangular, with large cone-shaped process at apex, basal arch high, basal arm slender and curved, parameres separate narrower distance at base, cone-shaped apically, and the female subgenital plate pliers-shaped, without lateral process, with two spermathecae, oval, nearly equal. The new species is compared with the similar congener F. (Forcipomyia) lochmocola Zou and Yu, 1991. We provide separate keys for identification of the males and females of the species of subgenus F. (Forcipomyia) Meigen in China.

The Immatures of Culicoides trilineatus (Diptera: Ceratopogonidae) Potential Vector of the Bluetongue Virus.

The fourth instar larva and pupa of Culicoides trilineatus Fox (Diptera, Ceratopogonidae), a species considered as potential vector of the bluetongue virus in Central and South America, are described, illustrated, and photomicrographed for the first time by using binocular, phase-contrast, and scanning electron microscopy. The immatures were collected by using a siphon bottle in tree holes in Salta Province, Argentina, transported to the laboratory, and there reared to the adult's emergence. They are compared with the immatures of Culicoides debilipalpis Lutz (Diptera, Ceratopogonidae), another Neotropical species that develops in tree holes. Details on larval biology and habitat are given.

Egg and Larval Morphology of Culicoides sonorensis (Diptera: Ceratopogonidae).

The egg and larval instars (L1-L4) of Culicoides sonorensis Wirth and Jones are described. Average (range) of body lengths of a laboratory colony (Van Ryn) were 0.45 (0.43-0.48) mm for eggs, 0.72 (0.39-0.92) mm for L1, 1.8 (1.3-2.2) mm for L2, 3.6 (2.2-5.0) mm for L3, and 5.9 (5.0-6.9) mm for L4. Head capsule lengths were 0.067 (0.064-0.070) mm for L1, 0.100 (0.094-0.110) for L2, 0.157 (0.144-0.168) for L3, and 0.316 (0.300-0.324) mm for L4. Wild and colony L4 larvae differed in many standard metrics such as head length or width, but head ratios and pharyngeal armature measurements were comparable. Standard external and internal features of the head capsule are described. Scanning electron microscope photos revealed pharyngeal armature structure was similar for L2-L4. The pharyngeal armature and piercing mandibles suggest a generalist feeder and opportunistic predator. Egg ansullae were randomly distributed (as opposed to rows in most other Culicoides spp.) and averaged 1.98 ansullae per 25 µm2 of egg surface. Ansullae were widened and porous apically, averaging 1.29 microns long, 0.65 µm at the base, and 1.06 µm at the apex. Only the L1 had a proleg, structurally complex, and featuring five rows of posteriorily directed, shorter spines (1-3 µm long) and 8-10 setae 18 µm long with hooked tips. On terrestrial mud, this proleg was used for surface crawling, and the L1 navigated dry conditions better than the more aquatic L2-L4 stages. Eggs are laid above waterline, and the L1 proleg is probably an advantage in terrestrial surface movement.

Description of fourth instar larva and pupa of Atrichopogon delpontei Cavalieri and Chiossone (Diptera: Ceratopogonidae) from Brazilian Amazonia.

The fourth instar larva and pupa of Atrichopogon delpontei Cavalieri and Chiossone are described for the first time. The immatures were collected from stream margins in the northern Brazilian states Rondônia and Piauí, and subsequently reared to adults. Larvae and pupae are illustrated and photomicrographed. Details on the rearing process and feeding behavior in laboratory, bionomics and notes on habitats are also provided.

Description of fourth instar larva and pupa of Atrichopogon delpontei Cavalieri and Chiossone (Diptera: Ceratopogonidae) from Brazilian Amazonia

ABSTRACT The fourth instar larva and pupa of Atrichopogon delpontei Cavalieri and Chiossone are described for the first time. The immatures were collected from stream margins in the northern Brazilian states Rondônia and Piauí, and subsequently reared to adults. Larvae and pupae are illustrated and photomicrographed. Details on the rearing process and feeding behavior in laboratory, bionomics and notes on habitats are also provided.

Atypical mechanoreceptors in larvae of biting midges Forcipomyia nigra (Diptera: Ceratopogonidae).

The dorsolateral setae of Forcipomyia nigra have been investigated. They are mechanoreceptors, sensilla trichoidea, innerved by a single neuron. The tubular body is located at the base of the hair shaft. Although the fine structure of the dorsolateral setae is similar to dorsal setae that perform a double function: secretion of a hygroscopic viscous substance through the pores and mechanoreception dorsolateral setae, they do not secrete any fluid. In both types of setae, trichogen cells (hair forming cells) produce the hair shafts and thereafter do not retract from the cavity of the setae. They contain a large polyploid nucleus and expanded bundles of microtubules. In dorsolateral mechanoreceptors, the microtubules form a network around the nucleus of the trichogen cell and are especially numerous in the cytoplasm invading the interior of the seta, which is evidenced by immunofluorescence light microscopy. No tormogen cell, responsible for the production of the setal socket, was found. Our observations indicate that the dorsolateral setae are solely mechanoreceptors but their trichogen cells reveal some glandular activity.

Description of the last instar larva and new contributions to the knowledge of the pupa of Dasyhelea mediomunda Minaya (Diptera, Culicomorpha, Ceratopogonidae).

The fourth instar larva of Dasyhelea mediomunda Minaya is described for the first time and a complete description of the pupa is provided, through use of phase-contrast microscope and scanning electron microscope. Studied specimens were collected in a pond connected to a small wetland "mallin" on the Patagonian steppe, Chubut province, Argentina.

Metamorphic changes in abdominal spines of Forcipomyia nigra pupae (Diptera: Ceratopogonidae).

Pupae of Forcipomyia nigra biting midges bear double rows of dorsal and lateral spines. Their arrangement corresponds to the distribution of larval mechanosensory setae. They are serrated simple cuticular structures with tubercles but, in contrast to larval secretory mechanoreceptors, they are not innervated and do not exhibit any pores. The ultrastructure of abdominal spines varies among different pupal stages. They are produced by epidermal cells which fill the interior of the spine. In the youngest pupae epidermal cells are tightly packed and adhere to the cuticle. Then, the cells withdraw from the spinal cavity and the beginning of autophagy is observed. The last stage represents abdominal spines without any cellular material and then apoptosis probably proceeds in the withdrawn epidermal cells. Since the pupal spines occupied the same region of the segment as the larval setae, we consider that the same genes are responsible for their formation as for the formation of epidermal cells but that their mechanosensory and secretory function is no longer needed.

Ultrastructure of mediodorsal setae in biting midge larvae of the genus Atrichopogon Kieffer with notes on their biological significance (Diptera: Ceratopogonidae).

The ultrastructure of the strong mediodorsal setae in terrestrial stage IV larvae of Atrichopogon (Meloehelea) oedemerarum and A. (M.) meloesugans was examined using light, scanning and transmission electron microscopy. Serrated setae placed on prominent processes are distributed in pairs on all thoracic and abdominal segments. Setae are innervated by a single dendrite and their surface has no pores. The trichogen cell is not retracted from the setal lumen on completion of the hair-forming process but fills the mediodorsal seta also when the larval cuticle is fully sclerotised. Such a phenomenon was previously reported in terrestrial larvae of the genus Forcipomyia. We suggest that the mediodorsal setae described in Atrichopogon are plesiotypic mechanoreceptors for the subfamily Forcipomyiinae. They are preserved in the truly terrestrial larvae of Atrichopogon, but modified to secretory setae in the genus Forcipomyia. Both genera bearing distinct mediodorsal setae have developed functional tracheal gills, unknown in other biting midges.

Ultrastructural studies on the midgut of biting midge Forcipomyia nigra (Winnertz) (Diptera: Ceratopogonidae).

Biting midges belonging to the genus Forcipomyia are known to be hematophagous, predatory or saprophagous. Different stages of Forcipomyia nigra midges were investigated to provide a description of midgut ultrastructure. Larvae feeding on decaying organic matter possess simple, straight alimentary tracts whose middle regions are the longest. TEM studies of the larval midgut epithelium reveal that digestive cells show different ultrastructure depending on their age. The older cells with electron-dense cytoplasm degenerate while the younger ones with electron-lucent cytoplasm remain active in digestion. In saprophagous females, the ultrastructure of midgut epithelium changes according to the age of flies. Oogenesis induces degeneration of digestive cells and utilization of reserve material accumulated by them. The midgut epithelia of male midges consist of digestive and regenerative cells that show no evidence of cell degeneration as observed in females. Our results demonstrate differences between midgut digestive cells of males and females.

A new species of Forcipomyia Meigen (Diptera: Ceratopogonidae) described with immature stages from India.

Life stages of Forcipomyia parasecuris sp. n. is described, illustrated and photomicrograph based on examination with scanning electron microscope and binocular microscope. The larvae were collected from rotting banana stump of a banana garden at lower Gangetic plain area in India. Absence of ocular sensillum and campaniform sensillum in ocular area of pupa, a synapomorphic character of this proposed new species exhibits its uniqueness in genus Forcipomyia. This is also the first life stage of any Forcipomyia described from India.

Sensilla coeloconica ringed by microtrichia in host-seeking biting midges.

The distribution and morphology of antennal sensilla coeloconica in parasitic and predaceous biting midges were studied in females of Forcipomyia (feeding on the blood of frogs), Atrichopogon (feeding on haemolymph), Austroconops, Culicoides (feeding on the blood of birds and mammals) and Brachypogon (feeding on haemolymph and dissolved tissues of insects) (all: Diptera: Ceratopogonidae). A Lower Cretaceous female of Archiculicoides (Diptera: Ceratopogonidae) from Lebanese amber, which fed on the blood of unknown vertebrates, was also examined. In sensilla coeloconica ringed by microtrichia, the peg is grooved longitudinally and protrudes distinctly from the pit. We suggest that the microtrichia encircling the protruding peg form a structure resembling a picket fence in order to maintain a higher level of humidity, which facilitates the capture and transport of odour molecules through the channels in the peg wall. Sensilla coeloconica ringed by microtrichia function as very effective chemoreceptors in host- and prey-seeking activity. During the evolution of Ceratopogonidae, sensilla coeloconica with a fence of microtrichia have evolved twice in groups feeding on the blood of vertebrates (i.e. in the basal lineage: Lower Cretaceous or earlier) and in the subgenus Lasiohelea of Forcipomyia (Palaeogene). Sensilla coeloconica ringed by microtrichia are described for the first time in the relict genus Austroconops.

Comparative study of antennal and maxillary palp olfactory sensilla of female biting midges (Diptera: Ceratopogonidae: Culicoides) in the context of host preference and phylogeny.

Culicoides biting midges (Diptera Ceratopogonidae) are vectors of disease, including bluetongue and African horse sickness. Host preference of these insects is primarily regulated by olfactory cues, detected by olfactory sensilla on the antennae and maxillary palps. In this study, we analyzed the sensillum repertoire of biting midge species with known host preferences. Five different morphological sensillum types, sensilla trichodea, s. chaetica, s. ampullacea, s. coeloconica, and grooved peg sensilla, were present on the antennae of all species. In addition sensilla basiconica were present on the maxillary palps. We found that the numbers of short blunt-tipped s. trichodea, s. coeloconica, and s. basiconica are significantly higher in the ornithophilic Culicoides festivipennis (Kieffer) compared with the mammalophilic Culicoides obsoletus (Meigen) and Culicoides chiopterus (Meigen). In contrast, we found that the mammalophilic Culicoides pulicaris (L.) and the opportunistic Culicoides punctatus (Meigen) have intermediate numbers of these sensillum types. Comparison with available data from other species strongly suggests that these differences in the number of specific sensillum types, in general, are a reflection of host preference and not of phylogeny. We discuss the putative function of the individual sensillum types in relation to host volatile detection.

Composition and antimicrobial activity of fatty acids detected in the hygroscopic secretion collected from the secretory setae of larvae of the biting midge Forcipomyia nigra (Diptera: Ceratopogonidae).

The hygroscopic secretion produced by the secretory setae of terrestrial larvae of the biting midge Forcipomyia nigra (Winnertz) was analysed using gas chromatography coupled with mass spectrometry (GC-MS). The viscous secretion is stored at the top of each seta and absorbs water from moist air. GC-MS analyses (four independent tests) showed that the secretion contained 12 free fatty acids, the most abundant of which were oleic (18:1), palmitic (16:0), palmitoleic (16:1) and linoleic (18:2). Other acids identified were valeric (5:0), enanthic (7:0), caprylic (8:0), pelargonic (9:0), capric (10:0), lauric (12:0), myristic (14:0) and stearic (18:0). Two other compounds, glycerol and pyroglutamic acid, were also found. The antibacterial activity of the fatty acids and pyroglutamic acid was tested using the agar disc diffusion method and targeted Gram positive (Bacillus cereus, Bacillus subtilis, Enterococcus faecalis) and Gram negative bacterial strains (Citrobacter freundii, Pseudomonas aeruginosa, Pseudomonas fluorescens). The antifungal activity was tested by determining minimal inhibitory concentration (MIC) of examined compounds. Fatty acids were tested against enthomopathogenic fungi (Paecilomyces lilacinus, Paecilomyces fumosoroseus, Lecanicillium lecanii, Metarhizium anisopliae, Beauveria bassiana (Tve-N39), Beauveria bassiana (Dv-1/07)). The most effective acids against bacterial and fungal growth were C(9:0), C(10:0) and C(16:1), whereas C(14:0), C(16:0,) C(18:0) and C(18:1) demonstrated rather poor antifungal activity and did not inhibit the growth of bacteria. The antimicrobial assay investigated mixtures of fatty and pyroglutamic acids (corresponding to the results of each GC-MS test): they were found to be active against almost all the bacteria except P. fluorescens and also demonstrated certain fungistatic activity against enthomopathogenic fungi. The hygroscopic secretion facilitates cuticular respiration and plays an important role in the antimicrobial protection of F. nigra larvae living in moist terrestrial habitats.

Morphology and histology of secretory setae in terrestrial larvae of biting midges of the genus Forcipomyia (Diptera: Ceratopogonidae).

Apneustic larvae of the genus Forcipomyia possess unique secretory setae located on the dorsal surface along the body in two rows, one pair on each thoracic and abdominal segment and two pairs on the head. Morphological and histological studies of secretory setae in fourth instar larvae of Forcipomyia nigra (Winnertz) and Forcipomyia nigrans Remm indicate they are modified mechanoreceptors (sensilla trichodea) in which the trichogen cell is a glandular cell producing a hygroscopic secretion. The cytoplasm of the glandular trichogen cell fills the lumen of a secretory seta, which shows one or more pores on the apex. The cytoplasm contains numerous microtubules responsible for transportation of proteinaceous vesicles, and an extremely large polyploid nucleus typical of gland cells. The main role of the hygroscopic secretion is to moist the body and thus facilitate cuticular respiration.

Patterning of a compound eye on an extinct dipteran wing.

We have discovered unexpected similarities between a novel and characteristic wing organ in an extinct biting midge from Baltic amber, Eohelea petrunkevitchi, and the surface of a dipteran's compound eye. Scanning electron microscope images now reveal vestigial mechanoreceptors between the facets of the organ. We interpret Eohelea's wing organ as the blending of these two developmental systems: the formation and patterning of the cuticle in the eye and of the wing. Typically, only females in the genus carry this distinctive, highly organized structure. Two species were studied (E. petrunkevitchi and E. sinuosa), and the structure differs in form between them. We examine Eohelea's wing structures for modes of fabrication, material properties and biological functions, and the effective ecological environment in which these midges lived. We argue that the current view of the wing organ's function in stridulation has been misconstrued since it was described half a century ago.

Sperm ultrastructure in Chironomoidea (Insecta, Diptera).

The fine structure of spermatozoa from several species of chironomids, of Culicoides sp. (Ceratopogonidae) and of Odagmia pontina (Simulidae) was studied. A synapomorphic feature, consisting of nine kidney-shaped structures forming the centriole adjunct, was found in the chironomid species. All members of Chironomoidea share a mono-layered acrosome and a flagellar axoneme, provided with accessory tubules with 15 protofilaments in their tubular wall. The axoneme has a 9+9+2 pattern, but in an unidentified species of chironomid, a 9+9+0 model was observed where the central complex and the spokes are missing. Sperm motility is, however, maintained in all the examined species. The spermatozoa of this taxon have the tendency to complete maturation during their progression along the deferent ducts. Thus, in the proximal region of these ducts, they often show remnants of the spermatid cytoplasm.

Sergeia podlipaevi gen. nov., sp. nov. (Trypanosomatidae, Kinetoplastida), a parasite of biting midges (Ceratopogonidae, Diptera).

Three strains of a trypanosomatid protozoan were isolated from the midguts of two naturally infected species of biting midges [Culicoides (Oecacta) festivipennis and Culicoides (Oecacta) truncorum] and characterized by light and electron microscopy and by molecular techniques. Morphological characteristics and sequences of the 18S rRNA, 5S rRNA, spliced leader RNA and glycosomal glyceraldehyde-3-phosphate dehydrogenase genes indicate that the studied flagellates represent a novel phylogenetic lineage within the Trypanosomatidae. Based on phylogenetic analyses, the novel endosymbiont-free, monoxenous trypanosomatid was classified as Sergeia podlipaevi gen. nov., sp. nov. Interestingly, it is closely related to another trypanosomatid species that parasitizes the sand fly Lutzomyia evansi, a blood-sucking dipteran from South America. The type strain of S. podlipaevi sp. nov., ICUL/CZ/2000/CER3, was obtained from Malpighian tubes. Of 2518 females of seven species of biting midges trapped in the Czech Republic, more than 1.5 % were infected by trypanosomatid parasites. An unrelated insect species, Culicoides (Monoculicoides) nubeculosus, was experimentally infected with S. podlipaevi, demonstrating that its host range extends to different subgenera of biting midges.

[Formation of the peritrophic membrane in the gut of Colicoides punctatus (Diptera: Ceratopogonidae)]. / Obrazovanie peritrofitseskoi membrany v kishechnike Culicoides punctatus (Diptera: Ceratopogonidae).

Using light and electron microscopy, the structure of the peritrophic membrane (PM) was studied in females of the biting midge Culicoides punctatus (Mg.) during the process of blood meal digestion. The PM formation occurs in the posterior part of midgut and lasts during the most time of the digestive cycle. The PM precursors are probably not associated with any intracellular granules. The PM consists of two main components: light structural component and dark amorphous matrix, both of which are directly released from the entire microvillar surface. The aggregation of secreted components takes place in the gut lumen to form gel-like multilayered PM up to 6 microns thickness with bundles of microfibrills situated in the PM surface facing the lumen. Similarities and differences of the PM formation in most groups of blood-sucking insects are discussed.