Natural infection with Leishmania (Mundinia) martiniquensis supports Culicoides peregrinus (Diptera: Ceratopogonidae) as a potential vector of leishmaniasis and characterization of a Crithidia sp. isolated from the midges.

The prevalence of autochthonous leishmaniasis in Thailand is increasing but the natural vectors that are responsible for transmission remain unknown. Experimental in vivo infections in Culicoides spp. with Leishmania (Mundinia) martiniquensis and Leishmania (Mundinia) orientalis, the major causative pathogens in Thailand, have demonstrated that biting midges can act as competent vectors. Therefore, the isolation and detection of Leishmania and other trypanosomatids were performed in biting midges collected at a field site in an endemic area of leishmaniasis in Tha Ruea and a mixed farm of chickens, goats, and cattle in Khuan Phang, Nakhon Si Thammarat province, southern Thailand. Results showed that Culicoides peregrinus was the abundant species (>84%) found in both locations and only cow blood DNA was detected in engorged females. Microscopic examination revealed various forms of Leishmania promastigotes in the foregut of several C. peregrinus in the absence of bloodmeal remnants, indicating established infections. Molecular identification using ITS1 and 3'UTR HSP70 type I markers showed that the Leishmania parasites found in the midges were L. martiniquensis. The infection rate of L. martiniquensis in the collected flies was 2% in Tha Ruea and 6% in Khuan Phang, but no L. orientalis DNA or parasites were found. Additionally, organisms from two different clades of Crithidia, both possibly new species, were identified using SSU rRNA and gGAPDH genes. Choanomastigotes and promastigotes of both Crithidia spp. were observed in the hindgut of the dissected C. peregrinus. Interestingly, midges infected with both L. martiniquensis and Crithidia were found. Moreover, four strains of Crithidia from one of the clades were successfully isolated into culture. These parasites could grow at 37°C in the culture and infect BALB/c mice macrophages but no multiplication was observed, suggesting they are thermotolerant monoxenous trypanosomatids similar to Cr. thermophila. These findings provide the first evidence of natural infection of L. martiniquensis in C. peregrinus supporting it as a potential vector of L. martiniquensis.

Antennal morphology and sensilla ultrastructure of the malaria vectors, Anopheles maculatus and An. sawadwongporni (Diptera: Culicidae).

Mosquitoes rely mainly on the olfactory system to track hosts. Sensilla contain olfactory neuron receptors that perceive different kinds of odorants and transfer crucial information regarding the surrounding environment. Anopheles maculatus and An. sawadwongporni, members of the Maculatus Group, are regarded as vectors of malaria in Thailand. The fine structure of their sensilla has yet to be identified. Herein, scanning electron microscopy is used to examine the sensilla located on the antennae of adults An. maculatus and An. sawadwongporni, collected from the Thai-Myanmar border. Four major types of antennal sensilla are discovered in both species: chaetica, coeloconica, basiconica (grooved pegs) and trichodea. The antennae of female An. maculatus have longer lengths (µm, mean ± SE) in the long sharp-tipped trichodea (40.62 ± 0.35 > 38.20 ± 0.36), blunt-tipped trichodea (20.39 ± 0.62 > 18.62 ± 0.35), and basiconica (7.84 ± 0.15 > 7.41 ± 0.12) than those of An. sawadwongporni. Using light microscopy, it is found that the mean numbers of large sensilla coeloconica (lco) on both flagella in An. maculatus (left: 32.97 ± 0.48; right: 33.27 ± 0.65) are also greater when compared to An. sawadwongporni (left: 30.40 ± 0.62; right: 29.97 ± 0.49). The mean counts of lco located on flagellomeres 1-3, 6, and 9 in An. maculatus are significantly higher than those of An. sawadwongporni. The data in this study indicate that two closely related Anopheles species exhibit similar morphology of sensilla types, but show variations in length, and likewise in the number of large sensilla coeloconica between them, suggesting they might be causative factors that affect their behaviors driven by the sense of smell.

Ultrastructure of sensilla on the antennae and maxillary palpi of the human-biting black flies, Simulium nigrogilvum and Simulium umphangense, (Diptera: Simuliidae) in Thailand.

Antennae and maxillary palpi are the most important sensory organs involved in the behaviors of black flies. The ultrastructure of sensilla on these sensory appendages of two human-biting black fly species, Similium nigrogilvum and Simulium umphangense, was studied for the first time. Wild adult females of both species were collected in Umphang District, Tak Province, western Thailand. The morphology and distribution of sensilla were examined using scanning electron microscopy. Overall, the morphology of the antennae and maxillary palpi and distribution of sensilla are similar in the two species. Four major types of sensilla were found on the antennae of both species: sensilla basiconica (three subtypes), coeloconica, chaetica (four subtypes), and trichodea. However, sensilla basiconica subtype IV are only present on the antennal surface of S. nigrogilvum. Sensilla trichodea are the most abundant among the four types of sensilla that occur on the antennae of both species. Significant differences in the length of the antennae (scape and flagellomere IX), length of the maxillary palpi (whole and palpal segments I, III, IV and V), and the length and basal width of four sensilla types (trichodea, chaetica, basiconica, and coeloconica) were found. In addition, two types of sensilla were observed on the maxillary palpi: sensilla chaetica (three subtypes) and bulb-shaped sensilla. Differences were observed in the numbers of bulb-shaped sensilla in the sensory vesicles of S. nigrogilvum and S. umphangense. The findings are compared with the sensilla of other insects, and the probable functions of each sensillum type are discussed. The anatomical data on sensory organs derived from this study will help to better understand black fly behavior.

Significance of eggshell morphology as an additional tool to distinguish species of sand flies (Diptera: Psychodidae: Phlebotominae).

Morphological characteristics of eggshells are important in sand fly ootaxonomy. In this study, eggshells from Phlebotomus stantoni Newstead, Sergentomyia khawi (Raynal), and Grassomyia indica (Theodor) sand flies collected in Chiang Mai province, Thailand were examined and characterized using light microscopy (LM) and scanning electron microscopy (SEM). Then, eggshell morphology of these three species was described for the first time. Each gravid female was forced to lay eggs by decapitation and the eggs were collected for SEM analysis. Egg laying females were identified by morphological examination and molecular typing using cytochrome b (Cytb) as a molecular marker. The chorionic sculpturing of Ph. stantoni eggs combines two patterns on the same egg: unconnected parallel ridges and reticular patterns. Sergentomyia khawi and Gr. indica have similar chorionic polygonal patterns, but their exochorionic morphology and aeropylar area are different. Results indicate that eggshell morphological characteristics such as chorionic pattern, exochorionic morphology, inter-ridge/boundary area, aeropylar area (including the number of aeropyles) and basal layer, can be useful to develop morphological identification keys of eggs. These can serve as an additional tool to distinguish species of sand flies. In addition, the chorionic sculpturing of the eggs of the three species of sand flies observed by LM is useful for species identification in gravid females with spermathecae obscured by eggs.

UV-C radiation during the pupal stage affects morphological changes of wings in Tribolium castaneum (Col; Tenebrionidae).

Purpose: To reveal the effects of Ultraviolet-C (UV-C) on the elytra and hindwing morphology of Tribolium castaneum. Material and methods: Zero-day-old-pupae were irradiated with UV-C at a distance of 35 cm for 1, 2, 4, 8, 16, 32, or 64 min. Changes in wing morphologies were examined using light and scanning electron microscope. Results: UV-C radiation decreased the adult emergence rate and the insect body mass. Morphological changes of the elytra and hindwings in the adults were classified into nine grades. The treated insects had wrinkled and split elytra, and hindwings were not folded properly. Radiation altered the size of elytra, hindwings and wing shape. An analysis of the color intensity indicated that the irradiated beetles had darker elytra. The veins of hindwings became darker, while the membranous area had a lighter color than the control. UV-C radiation also affected the thickness of the elytra. Scanning electron microscopy revealed that UV-C caused deformity of elytra surface and decreased the number of hair sensilla. Conclusions: Results indicate that the elytra and hindwing morphology were altered by UV-C radiation. However, further analysis is required to evaluate the response of T. castaneum to UV-C radiation at the gene level.

Morphology of immature blow fly Hypopygiopsis infumata (Bigot) (Diptera: Calliphoridae), a potential species of forensic importance.

Blow flies of the genus Hypopygiopsis are forensically-important, as their larvae are commonly associated with human corpses. Within a forensic entomology context, species identification of specimens collected from human corpses is the initial mandatory step in the investigation. Without identification, complete interpretation of entomological evidence is challenged. In this study, the ultrastructures of eggs, all instars, and puparia of Hypopygiopsis infumata (Bigot) are presented based on assessment with scanning electron microscopy (SEM) and light microscopy (LM). Distinctive features used for species identification of all stages are highlighted. Eggs have a slightly widening median area extending almost the entire length. Larvae are vermiform-shaped, creamy white, and have a smooth integument. The pseudocephalon of larvae bears sensory structures (i.e., antennal dome, maxillary palpus and ventral organ). In the first instar, two tufts of cirri are observed along the dorsal margin of the mouth opening. In the second and third instars, six minute tubercles are present along the peripheral rims of the last abdominal segment. The anterior spiracle of the second, third instar, and puparia is fan-shape of single row, comprising 9-11 papillae. The cuticular spines between the 1st and 2nd thoracic segments of the third instar possess many rows of posteriorly-projecting acuminate spines in clusters. In puparia, at the latero-dorsal edge of the 1st abdominal segment, a cluster of ∼92 bubble membranes is present in young puparia (20-24 h). The peristigmatic tufts adjacent to the posterior spiracle of the second instar, third instar, and puparia are heavily branches of long, fine hairs. Our results demonstrate the morphology of eggs, larvae, and puparia of H. infumata are similar to other species in Hypopygiopsis. This study highlighted the main features of cephaloskeleton of H. infumata larvae as observed under LM. Particular attention is given to oral sclerite and rough surface of dorsal cornua which can distinguish between H. infumata and H. tumrasvini.

An effective method for the identification and separation of Anopheles minimus, the primary malaria vector in Thailand, and its sister species Anopheles harrisoni, with a comparison of their mating behaviors.

BACKGROUND: Species of the Anopheles minimus complex are considered to be the primary vectors of malaria in South and Southeast Asia. Two species of the complex, Anopheles minimus and Anopheles harrisoni, occur in Thailand. They are sympatric and difficult to accurately distinguish based on morphological characters. The aim of this study was to investigate the potential of antennal sensory organs to distinguish these two species. Additionally, we investigated their ability to mate in cages of different sizes, as well as the possible mechanism(s) that evokes stenogamous behavior. METHODS: Large sensilla coeloconica present on the antennae of females of An. minimus and An. harrisoni were counted under a conventional light microscope and various types of antennal sensilla were examined under a scanning electron microscope (SEM). Determinations of mating ability were carried out in 20 and 30 cm3 cages with a density resting surface (DRS) of 7.2. The insemination rate, frequency of clasper (gonocoxopodite) movement of the male genitalia during induced copulation and duration of mating of the two species were compared. RESULTS: The mean numbers of large sensilla coeloconica on antennal flagellomeres 1-8 and the mean number of large sensilla coeloconica on each flagellum in An. minimus (26.25) and An. harrisoni (31.98) were significantly different. Females of both species bear five types of antennal sensilla: chaetica, trichodea, basiconica, coeloconica and ampullacea. Marked differences in the structure of the large sensilla coeloconica were observed between the two species. Furthermore, only An. minimus could copulate naturally in the small cages. The frequency of clasper movement in the stenogamous An. minimus was significantly higher than in An. harrisoni, but there was no difference in the duration of mating. CONCLUSIONS: To our knowledge, this study is the first to examine and discover the usefulness of large sensilla coeloconica on the antennae of females and the frequency of clasper movement in males for distinguishing the sibling species An. minimus and An. harrisoni. The discovery provides an effective and relatively inexpensive method for their identification. Additionally, the greater frequency of clasper movement of An. minimus might influence its ability to mate in small spaces.

Scanning electron microscopy of antennal sensilla of the eight Anopheles species of the Hyrcanus Group (Diptera: Culicidae) in Thailand.

Antennal sensilla were first investigated in the eight medically and veterinary important Anopheles mosquito species (Anopheles argyropus, Anopheles crawfordi, Anopheles nigerrimus, Anopheles nitidus, Anopheles paraliae (= Anopheles lesteri), Anopheles peditaeniatus, Anopheles pursati, and Anopheles sinensis) of the Hyrcanus Group in Thailand, using scanning electron microscopy (SEM). Four types of sensilla, including sensilla chaetica (large and small), sensilla trichodea (sharp- and blunt-tipped), sensilla basiconica or grooved pegs (types I, II, and III), and sensilla coeloconica (large and small), were observed on the female antennae of the eight species. The greatest number of sensilla found along the flagellum of all the Anopheles species consisted of sensilla trichodea. Grooved pegs type II were not found on the antennae of An. peditaeniatus. Interestingly, clusters of 10-15 grooved pegs type III, with blunt-tipped and unevenly grooved-lengthwise sensilla, and a sunken group of 7-12 grooved pegs type III, with slightly curved and point-tipped sensilla, were found distally on flagellomeres 3-7 of An. argyropus and An. peditaeniatus, respectively. In addition, the key for species identification, based on fine structure and morphometrics of antennal sensilla among the eight species, was constructed and differentiated successfully. However, in order to focus intensively on the exact function of these sensilla, further electrophysiological study is needed in understanding their significant role in mosquito behavior, especially when these insects seek hosts for transmitting pathogens to humans.

Generation and preclinical immunogenicity study of dengue type 2 virus-like particles derived from stably transfected mosquito cells.

Recent phase IIb/III trials of a tetravalent live attenuated vaccine candidate revealed a need for improvement in the stimulation of protective immunity against diseases caused by dengue type 2 virus (DENV-2). Our attempts to develop particulate antigens for possibly supplementing live attenuated virus preparation involve generation and purification of recombinant DENV-2 virus-like particles (VLPs) derived from stably (prM+E)-expressing mosquito cells. Two VLP preparations generated with either negligible or enhanced prM cleavage exhibited different proportions of spherical particles and tubular particles of variable lengths. In BALB/c mice, VLPs were moderately immunogenic, requiring adjuvants for the induction of strong virus neutralizing antibody responses. VLPs with enhanced prM cleavage induced higher levels of neutralizing antibody than those without, but the stimulatory activity of both VLPs was similar in the presence of adjuvants. Comparison of EDIII-binding antibodies in mice following two adjuvanted doses of these VLPs revealed subtle differences in the stimulation of anti-EDIII binding antibodies. In cynomolgus macaques, VLPs with enhanced prM cleavage augmented strongly neutralizing antibody and EDIII-binding antibody responses in live attenuated virus-primed recipients, suggesting that these DENV-2 VLPs may be useful as the boosting antigen in prime-boost immunization. As the levels of neutralizing antibody induced in macaques with the prime-boost immunization were comparable to those infected with wild type virus, this virus-prime VLP-boost regimen may provide an immunization platform in which a need for robust neutralizing antibody response in the protection against DENV-2-associated illnesses could be tested.

An optimized expression vector for improving the yield of dengue virus-like particles from transfected insect cells.

Recombinant virus-like particles (rVLPs) of flaviviruses are non-infectious particles released from cells expressing the envelope glycoproteins prM and E. Dengue virus rVLPs are recognized as a potential vaccine candidate, but large scale production of these particles is hindered by low yields and the occurrence of cytopathic effects. In an approach to improve the yield of rVLPs from transfected insect cells, several components of a dengue serotype 2 virus prM+E expression cassette were modified and the effect of these modifications was assessed during transient expression. Enhancement of extracellular rVLP levels by simultaneous substitutions of the prM signal peptide and the stem-anchor region of E with homologous cellular and viral counterparts, respectively, was further augmented by codon optimization. Extensive formation of multinucleated cells following transfection with the codon-optimized expression cassette was abrogated by introducing an E fusion loop mutation. This mutation also helped restore the extracellular E levels affected negatively by alteration of a charged residue at the pr-M junction, which was intended to promote maturation of rVLPs during export. Optimized expression cassettes generated in this multiple add-on modification approach should be useful in the generation of stably expressing clones and production of dengue virus rVLPs for immunogenicity studies.

Scanning electron microscopy of Anopheles hyrcanus group (Diptera: Culicidae) eggs in Thailand and an ultrastructural key for species identification.

The eggs of Anopheles argyropus, Anopheles crawfordi, Anopheles nigerrimus, Anopheles nitidus, Anopheles paraliae, Anopheles peditaeniatus, Anopheles pursati, and Anopheles sinensis are described with the aid of scanning electron micrographs. Comparisons of the egg structure among the eight species showed that the eggs differed with respect to the following characteristics: the deck-complete (An. argyropus, An. nigerrimus, An. paraliae, An. peditaeniatus, and An. sinensis); variable (complete, split and incomplete decks found together within an egg batch/An. crawfordi); and division into an area at each end (An. nitidus and An. pursati). The ratios of the entire length per maximal deck width within the area covered by floats were 3.33-6.86 (An. sinensis), 8.78-18.20 (An. peditaeniatus), 13.67-22 (An. nigerrimus), 26.33-44.25 (An. paraliae), and 26.99-75.94 (An. argyropus). The numbers of float ribs were 21-27 (An. peditaeniatus) and 28-34 (An. nigerrimus), and the total numbers of anterior and posterior tubercles were 6-8 (An. paraliae) and 9-11 (An. argyropus). Exochorionic sculpturing was of reticulum type (An. argyropus, An. crawfordi, An. nigerrimus, An. nitidus, An. paraliae, An. peditaeniatus, and An. sinensis) and pure tubercle type (An. pursati). Attempts are proposed to construct a robust key for species identification based on the morphometrics and ultrastructures of eggs under scanning electron microscopy.

Larval fat body cells die during the early pupal stage in the frame of metamorphosis remodelation in Bombyx mori.

In holometabolus insects, morphology of the larval fat body is remodeled during metamorphosis. In higher Diptera, remodeling of the fat body is achieved by cell death of larval fat body cells and differentiation of the adult fat body from primordial cells. However, little is known about remodeling of the fat body at pupal metamorphosis in Lepidoptera. In this study, we found that cell death of the larval fat body in Bombyx mori occurs at shortly after pupation. About 30% of the fat body cells underwent cell death on days 1 and 2 after pupation. The cell death involved genomic DNA fragmentation, a characteristic of apoptosis. Surgical manipulation and in vitro culture of fat body cells revealed that 20-hydroxyecdysone and juvenile hormone had no effect on either initiation or progression of cell death. During cell death, a large increase in activity of caspase-3, a key enzyme of cell death, was observed. Western blot analysis of the active form of caspase-3-like protein revealed that the length of caspase-3 of B. mori was much larger than that of caspase-3 in other species. The results suggest that larval fat body cells of B. mori are removed through cell death, which is mediated by a caspase probably categorized in a novel family.