Integrating wing morphometrics and mitochondrial DNA analysis to assess the filaria vector Mansonia uniformis (Diptera: Culicidae) populations in Thailand.

Mansonia uniformis (Diptera: Culicidae) is recognized as a vector of Brugia malayi and has been reported to transmit Wuchereria bancrofti, both causing lymphatic filariasis in humans. This study employed geometric morphometrics (GM) to investigate wing shape variation and analyzed genetic diversity through cytochrome c oxidase subunit 1 (COI) gene analyses in Ma. uniformis populations across Thailand. Wing GM analyses indicated significant differences in wing shape based on Mahalanobis distances among nearly all population pairs (p < 0.05), with no significant correlation between wing shape and geographic distance (r = 0.210, p > 0.05). Genetic analyses identified 63 haplotypes and 49 polymorphic sites, with the overall population exhibiting a nucleotide diversity of 0.006 (± 0.001) and a haplotype diversity of 0.912 (± 0.017). Deviations from neutrality, as indicated by Tajima's D and Fu's FS tests for the overall Ma. uniformis populations in Thailand, were statistically significant and negative, suggesting population expansion (both p < 0.05). Analysis of molecular variance revealed no significant genetic structure when all populations were categorized based on collection sites and geographic regions. However, significant differences in FST values were observed between some populations. These findings enhance our understanding of the geographical and genetic factors influencing Ma. uniformis populations, which are crucial for developing effective control strategies in Thailand.

The Acquisition and Retention of Lumpy Skin Disease Virus by Blood-Feeding Insects Is Influenced by the Source of Virus, the Insect Body Part, and the Time since Feeding.

Lumpy skin disease virus (LSDV) is a poxvirus that causes severe systemic disease in cattle and is spread by mechanical arthropod-borne transmission. This study quantified the acquisition and retention of LSDV by four species of Diptera (Stomoxys calcitrans, Aedes aegypti, Culex quinquefasciatus, and Culicoides nubeculosus) from cutaneous lesions, normal skin, and blood from a clinically affected animal. The acquisition and retention of LSDV by Ae. aegypti from an artificial membrane feeding system was also examined. Mathematical models of the data were generated to identify the parameters which influence insect acquisition and retention of LSDV. For all four insect species, the probability of acquiring LSDV was substantially greater when feeding on a lesion compared with feeding on normal skin or blood from a clinically affected animal. After feeding on a skin lesion LSDV was retained on the proboscis for a similar length of time (around 9 days) for all four species and for a shorter time in the rest of the body, ranging from 2.2 to 6.4 days. Acquisition and retention of LSDV by Ae. aegypti after feeding on an artificial membrane feeding system that contained a high titer of LSDV was comparable to feeding on a skin lesion on a clinically affected animal, supporting the use of this laboratory model as a replacement for some animal studies. This work reveals that the cutaneous lesions of LSD provide the high-titer source required for acquisition of the virus by insects, thereby enabling the mechanical vector-borne transmission. IMPORTANCE Lumpy skin disease virus (LSDV) is a high consequence pathogen of cattle that is rapidly expanding its geographical boundaries into new regions such as Europe and Asia. This expansion is promoted by the mechanical transmission of the virus via hematogenous arthropods. This study quantifies the acquisition and retention of LSDV by four species of blood-feeding insects and reveals that the cutaneous lesions of LSD provide the high titer virus source necessary for virus acquisition by the insects. An artificial membrane feeding system containing a high titer of LSDV was shown to be comparable to a skin lesion on a clinically affected animal when used as a virus source. This promotes the use of these laboratory-based systems as replacements for some animal studies. Overall, this work advances our understanding of the mechanical vector-borne transmission of LSDV and provides evidence to support the design of more effective disease control programmes.

On the true identity of Sergentomyia gemmea and description of a closely related species: Se. raynali n. sp.

Several species of Leishmania are responsible for leishmaniases in Thailand, although little is known about their transmission. Sergentomyia gemmea has been suspected several times to transmit Leishmania martiniquensis. Some captures carried out in Thailand and Lao People's Democratic Republic have emphasized the scarcity of Se. gemmea, comprising only 1% of the collected females. The sequencing of cytochrome B mtDNA of our specimens showed that our specimens are not grouped with other Se. gemmea previously deposited in GenBank. The latter are grouped with some Se. khawi and Se. hivernus that we processed in the present study. We suspect misidentifications and propose focusing on the most useful characters for identification of Se. gemmea based on the examination of type-specimens. The examination of the ascoids exhibiting anterior spurs is the most important one. However, we also describe Se. raynali n. sp. exhibiting comparable spurs but differing from Se. gemmea by its original cibarium. Finally, the vectorial role of Se. gemmea appears very questionable in the absence of new evidence.

A review and illustrated description of Musca crassirostris, one of the most neglected haematophagous livestock flies.

Tabanids, stomoxyine flies, hippoboscids and tsetse flies are the most well-known brachyceran biting flies of livestock. Only a few other higher Diptera have developed the unique mouthparts required for blood feeding. These neglected blood feeders can also have direct effects on hosts through blood loss, and are likely to contribute to the transmission of pathogens. Musca crassirostris (Diptera: Muscidae) is one of the most abundant of the muscid flies with this haematophagous lifestyle; it is widespread in the Palaearctic, Afrotropical and Oriental regions. The present study reviews and summarizes the biology and morphology of this species, and its potential for impact on animals and humans. The study also provides a fully illustrated description of the fly to facilitate its identification, and reviews information on abundance, with a focus on recent trapping surveys in Thailand. When sampled using traps designed for other biting flies, M. crassirostris appears to be four and 45 times more abundant than stomoxyines and tabanids, respectively. High numbers of M. crassirostris in the vicinity of livestock have also been associated with outbreaks of disease, such as that of a fatal plague in bovine farms in Egypt. This calls for a reconsideration of its potential impacts on livestock economics and health, and thus the development of suitable control methods.

Morphology and DNA barcoding reveal three species in one: description of Culicoides cryptipulicaris sp. nov. and Culicoides quasipulicaris sp. nov. in the subgenus Culicoides.

Species of the genus Culicoides (Diptera: Ceratopogonidae) are well known for their importance in the field of medical and veterinary entomology. Culicoides spp. transmit a wide variety of pathogens, primarily viruses that affect animals and humans. In Europe, the most economically important disease transmitted by Culicoides is bluetongue (BT). Culicoides spp. have been recently involved as primary vectors for Schmallenberg disease. The taxonomy within the subgenus Culicoides has been historically difficult and reorganizations have been proposed regularly. The subgenus Culicoides includes species that are considered to be potential vectors for BT. High morphological intraspecific variability has been attributed to these species. This highlights the apparent presence of previously undetected cryptic species diversity in the subgenus. In the present study, a detailed morphological and molecular study of specimens belonging to Culicoides pulicaris s.l. and specimens resembling a cross between C. pulicaris and Culicoides punctatus revealed the presence of two new species: Culicoides cryptipulicaris and Culicoides quasipulicaris. Females of C. quasipulicaris and males of both species were morphologically distinguished from C. pulicaris (Linnaeus, 1758), whereas females of C. cryptipulicaris were identified using molecular techniques exclusively.

Olfactory Mechanisms for Discovery of Odorants to Reduce Insect-Host Contact.

Insects have developed highly sophisticated and sensitive olfactory systems to find animal or plant hosts for feeding. Some insects vector pathogens that cause diseases in hundreds of millions of people and destroy billions of dollars of food products every year. There is great interest, therefore, in understanding how the insect olfactory system can be manipulated to reduce their contact with hosts. Here, we review recent advances in our understanding of insect olfactory detection mechanisms, which may serve as a foundation for designing insect control programs based on manipulation of their behaviors by using odorants. Because every insect species has a unique set of olfactory receptors and olfactory-mediated behaviors, we focus primarily on general principles of odor detection that potentially apply to most insects. While these mechanisms have emerged from studies on model systems for study of insect olfaction, such as Drosophila melanogaster, they provide a foundation for discovery of odorants to repel vector insects or reduce their host-seeking behavior.

Origin, evolution and function of the hemipteran perimicrovillar membrane with emphasis on Reduviidae that transmit Chagas disease.

The peritrophic matrix is a chitin-protein structure that envelops the food bolus in the midgut of the majority of insects, but is absent in some groups which have, instead, an unusual extra-cellular lipoprotein membrane named the perimicrovillar membrane. The presence of the perimicrovillar membrane (PMM) allows these insects to exploit restricted ecological niches during all life stages. It is found only in some members of the superorder Paraneoptera and many of these species are of medical and economic importance. In this review we present an overview of the midgut and the digestive system of insects with an emphasis on the order Paraneoptera and differences found across phylogenetic groups. We discuss the importance of the PMM in Hemiptera and the apparent conservation of this structure among hemipteran groups, suggesting that the basic mechanism of PMM production is the same for different hemipteran species. We propose that the PMM is intimately involved in the interaction with parasites and as such should be a target for biological and chemical control of hemipteran insects of economic and medical importance.

Ecological and epidemiological status of species of the Phlebotomus perniciosus complex (Diptera: Psychodidae, Phlebotominae) in Morocco.

Leishmania infantum (Kinetoplastida: Trypanosomatidae) infection is transmitted by an infected female sand fly (Diptera: Psychodidae, Phlebotominae) of the subgenus Larroussius: Phlebotomus ariasi, Phlebotomus perniciosus, and Phlebotomus longicuspis in the Mediterranean basin. In Morocco, the vectorial role of P. ariasi was demonstrated, while that of P. longicuspis and P. perniciosus is not elucidated. In addition, Moroccan P. longicuspis and P. perniciosus populations present a higher morphologic and genetic variability. It was classified as P. perniciosus complex, including typical (PN) and atypical (PNA) morphs of P. perniciosus, P. longicuspis sensu stricto (LCss), and a sibling species of P. longicuspis (LCx). With the aim to study the ecological and epidemiological status of P. perniciosus complex species in Morocco, entomological surveys were carried out during three entomological seasons (2012, 2013, and 2014). We collected a total of 6298 specimens from 81 localities of northern, central, and southern Morocco. After describing the geographical distribution of P. perniciosus complex trough Morocco according to many variables (altitude, latitude, and longitude), we discuss the resulting epidemiological implications of its species. Our results highlight the geographical distribution of the two morphs of P. perniciosus through Morocco: PN is limited to the north, while PNA is widespread in northern, central, and southern Morocco. In terms of vectorial role, we hypothesize the potential involvement of PN, LCss, and LCx, at least, with P. ariasi, in the epidemiological cycle of L. infantum in Morocco.

Morphometric Analysis of Longipalpis (Diptera: Psychodidae) Complex Populations in Mato Grosso do Sul, Brazil.

Owing to the existence of cryptic species that are difficult to distinguish morphologically, the search for new taxonomic characters and methods for identifying and classifying sand flies continues. Lutzomyia longipalpis (Lutz & Neiva, 1912) and Lutzomyia cruzi (Mangabeira, 1938) (Diptera: Psychodidae) are two such species that occur in sympatry in some regions of Mato Grosso do Sul State (MS). Twenty females and twenty males from each of the five populations of Lu. longipalpis and one population of Lu. cruzi from MS were examined. An outlying population of Lu. longipalpis from Estrela de Alagoas, State of Alagoas, was used to compare the degree of divergence among the groups in MS. Specimens were cleared, mounted on slides, identified, and measured using LAS-Leica. The principal component analysis of morphometric characters showed a high degree of variation among females, while males varied to a lower degree. The populations of Alagoas and Miranda demonstrated the greatest variation. The first region, Alagoas, is geographically distant from the others and occurs under distinctly different ecological conditions, which likely accounts for the variation. Further studies should be made to elucidate the factors that contribute to the differences found between the populations of MS.

A New Species of Culicoides (Selfia) (Diptera: Ceratopogonidae) from Southeastern Utah.

A new species of biting midge, Culicoides (Selfia) moabensis, is described and illustrated from southeastern Utah. Its relationship to Culicoides (Selfia) multipunctatus Malloch and Culicoides (Selfia) brookmani Wirth is discussed, and modifications to existing keys to adult males and females of C. (Selfia) species are provided. Its abundance, seasonal distribution, and aspects of its reproductive and feeding biology and potential as an arbovirus vector are discussed.

Geometric morphometrics for the taxonomy of 11 species of Anopheles (Nyssorhynchus) mosquitoes.

The subgenus Anopheles (Nyssorhynchus) (Diptera: Culicidae) includes the primary vectors of Plasmodium spp. in Colombia. Most adult females of this subgenus are difficult to identify in the field using the available keys. With the objective of further investigating the discriminatory power of modern morphometrics, both landmark-based and outline-based approaches were explored using the wing venation geometry of 11 Anopheles (Nyssorhynchus) species. Wing shape was able to separate the closest species of the subgenus. When the 11 species were analysed together, validated classification scores on average 5.3-8.6 times higher than those expected by chance were observed. These scores computed from the total sample of 11 species were not satisfactory for the recognition of Anopheles benarrochi B, Anopheles oswaldoi s.l. and Anopheles strodei. These sibling species were captured in sympatry. To improve the identification power of the morphometric tool, it was necessary to analyse these species separately from the remaining species. The best classification scores were obtained using a combination of 12 landmarks collected not only on the intersections of wing veins, but also on spots. An outline approach also gave excellent reclassification scores. Another pair of sibling species, collected in allopatry, Anopheles nuneztovari and Anopheles rangeli, also showed high classification scores.

The mosquito Anopheles (Cellia) oreios sp. n., formerly species 6 of the Australasian Anopheles farauti complex, and a critical review of its biology and relation to disease.

Species 6 of the Australasian Anopheles farauti sibling species complex (Diptera: Culicidae) is described and formally named Anopheles oreios Bangs & Harbach, sp. n. Adult, pupal and fourth-instar larval specimens collected in the Baliem Valley, Papua Province, Indonesia, are characterized and compared with those of Anopheles farauti, Anopheles hinesorum, Anopheles irenicus and Anopheles torresiensis (formerly informally denoted as species 1, 2, 7 and 3, respectively). The variable wings of adult females, the male genitalia, the pupa and the fourth-instar larva of An. oreios are illustrated and DNA sequence data are included for regions coding for sections of the mitochondrial COI and COII genes. The biology of An. oreios and its relation to malaria transmission are discussed in detail and contrasted with the biology and disease relations of some members of the An. farauti and Anopheles punctulatus sibling species complexes.

Where do these bugs come from? Phenotypic structure of Triatoma infestans populations after control interventions in the Argentine Chaco.

House re-invasion by native triatomines after insecticide-based control campaigns represents a major threat for Chagas disease vector control. We conducted a longitudinal intervention study in a rural section (Area III, 407 houses) of Pampa del Indio, northeastern Argentina, and used wing geometric morphometry to compare pre-spray and post-spray (re-infestant bugs) Triatoma infestans populations. The community-wide spraying with pyrethroids reduced the prevalence of house infestation by T. infestans from 31.9% to < 1% during a four-year follow-up, unlike our previous studies in the neighbouring Area I. Two groups of bug collection sites differing in wing shape variables before interventions (including 221 adults from 11 domiciles) were used as a reference for assigning 44 post-spray adults. Wing shape variables from post-spray, high-density bug colonies and pre-spray groups were significantly different, suggesting that re-infestant insects had an external origin. Insects from one house differed strongly in wing shape variables from all other specimens. A further comparison between insects from both areas supported the existence of independent re-infestation processes within the same district. These results point to local heterogeneities in house re-infestation dynamics and emphasise the need to expand the geographic coverage of vector surveillance and control operations to the affected region.

Culex pipiens s.l. and Culex torrentium (Culicidae) in Wroclaw area (Poland): occurrence and breeding site preferences of mosquito vectors.

Both ornithophilic mosquito species, Culex pipiens s.l. (L.) and Culex torrentium (Martini, 1925), occur sympatric in temperate Europe. They are presumed to be primary vectors of West Nile and Sindbis viruses. Differentiation of these morphologically similar Culex species is essential for evaluation of different vector roles, for mosquito surveillance and integrated control strategies. Cx. torrentium has been neglected or erroneously determined as Cx. pipiens s.l. in some previous studies, because only males of both species can be diagnosed reliably by morphology. Thus, knowledge about species abundance, geographical distribution, breeding site preferences and the zoonotic risk assessment is incomplete also in Poland. In Wroclaw area (Silesian Lowland), besides typical urban breeding sites, huge sewage irrigation fields provide suitable breeding conditions for Culex species. They are also inhabited by 180 resident and migratory bird species serving as potential virus reservoirs. In this study, morphology of larvae and males as well as species diagnostic enzyme markers, namely adenylate kinase (AK) and 2-hydroxybutyrate dehydrogenase (HBDH), were used to discriminate Cx. pipiens s.l. and Cx. torrentium. In a total of 650 Culex larvae from 24 natural and artificial breeding sites, Cx. pipiens s.l. had a proportion of 94.0% and Cx. torrentium only 6.0%. It could be shown that both species are well adapted to various breeding site types like ditches, catch basins, flower pots and buckets with diverse water quality. Cx. torrentium preferred more artificial water containers in urban surrounding (12% species proportion), whereas in semi-natural breeding sites, Cx. torrentium was rare (3%). In 12 of 24 breeding sites, larvae of both species have been found associated.

Insects as vectors: systematics and biology.

Among the many complex relationships between insects and microorganisms such as viruses, bacteria and parasites, some have resulted in the establishment of biological systems within which the insects act as a biological vector for infectious agents. It is therefore advisable to understand the identity and biology of these vectors in depth, in order to define procedures for epidemiological surveillance and anti-vector control. The following are successively reviewed in this article: Anoplura (lice), Siphonaptera (fleas), Heteroptera (bugs: Cimicidae, Triatoma, Belostomatidae), Psychodidae (sandflies), Simuliidae (black flies), Ceratopogonidae (biting midges), Culicidae (mosquitoes), Tabanidae (horseflies) and Muscidae (tsetse flies, stable flies and pupipara). The authors provide a rapid overview of the morphology, systematics, development cycle and bio-ecology of each of these groups of vectors. Finally, their medical and veterinary importance is briefly reviewed.

Confirmation of Culex (Culex) tritaeniorhynchus summorosus (Diptera: Culicidae) as a separate species.

BACKGROUND & OBJECTIVES: Culex tritaeniorhynchus, a member of Cx. vishnui subgroup, is an important vector of Japanese encephalitis (JE) virus. Cx. tritaeniorhynchus summorosus considered as a variety or subspecies of Cx. tritaeniorhynchus, has been studied in detail to settle its taxonomic status. Surveys for the collection of Cx. tritaeniorhynchus from Chandigarh and adjoining areas have established the availability of Cx. summorosus from this area. METHODS: For the present investigation, collections have been made from Chandigarh and its adjoining areas (up to 60 km) for procuring the material. The detailed morphology including scanning electron microscopy of immatures (eggs and larvae) and adults of Cx. tritaeniorhynchus and Cx. summorosus has been studied and compared. Further, the interbreeding experiments of the two species were also conducted and efforts had been made to allow cross- breeding among the members of these two species. RESULTS: Comparison of egg, larval and adult morphology of Cx. summorosus with the parental species Cx. tritaeniorhynchus under the light and electron microscope, revealed significant differences. Moreover, these two species have also been found to be reproductively isolated as indicated by laboratory experiments. This settles the controversy on the status of Cx. summorosus and confirms its status as a distinct species. INTERPRETATION & CONCLUSION: The study establishes that the two species show considerable number of differences which are sufficient to consider them as separate species rather than subspecies or variant of Cx. tritaeniorhynchus. Furthermore, the absence of interbreeding between these two again confirms their separate specific status according to biological species concept. But, it is yet to ascertain whether Cx. summorosus is a vector of Japanese encephalitis like Cx. tritaeniorhynchus or not.

Morphological and molecular characterization of the ecological, biological and behavioural variants of the JE vector Culex tritaeniorhynchus: an assessment of its taxonomic status.

BACKGROUND & OBJECTIVES: Culex tritaeniorhynchus (Diptera: Culicidae), an important vector of Japanese encephalitis belongs to the Culex vishnui subgroup which includes two other vector species namely, Cx. Vishnui and Cx. pseudovishnui. Many varieties and types of Cx. tritaeniorhynchus have been reported, besides populations that exhibit behavioural and biological differences. This study was undertaken to find out whether Cx. tritaeniorhynchus populations exhibiting behavioural and biological variations, and those from different geographical areas, are comprised of more than one taxon or belong to a single taxon. METHODS: Morphological characterization was done by examining 153 morphological and morphometric characters in the larval (75), pupal (60) and adult stages (18) of five geographical populations of Cx. tritaeniorhynchus. Molecular characterization was done by PCR amplification of mitochondrial cytochrome c oxidase (COI) gene sequences (DNA barcodes) and another hypervariable genetic marker, the ribosomal DNA (16S). One-way ANOVA, principal component analysis (PCA) and discriminant factor analysis (DFA) were done for statistical analyses using the statistical package SPSS IBM version 19.0. RESULTS: Morphological characterization showed that no intraspecific differentiation can be made among the five geographical populations of Cx. tritaeniorhynchus. Molecular characterization done by DNA barcoding also showed that the COI sequences of all the five populations of Cx. tritaeniorhynchus grouped into a single taxonomic clade plus the genetic differentiation among these was non-significant and the overall gene flow among the populations was very high. Analysis of the ribosomal DNA also confirmed that the Cx. tritaeniorhynchus populations belonged to a single taxon. INTERPRETATION & CONCLUSION: Culex tritaeniorhynchus is a taxon that does not involve cryptic species.

Differential effects of inbreeding and selection on male reproductive phenotype associated with the colonization and laboratory maintenance of Anopheles gambiae.

BACKGROUND: Effective mating between laboratory-reared males and wild females is paramount to the success of vector control strategies aiming to decrease disease transmission via the release of sterile or genetically modified male mosquitoes. However mosquito colonization and laboratory maintenance have the potential to negatively affect male genotypic and phenotypic quality through inbreeding and selection, which in turn can decrease male mating competitiveness in the field. To date, very little is known about the impact of those evolutionary forces on the reproductive biology of mosquito colonies and how they ultimately affect male reproductive fitness. METHODS: Here several male reproductive physiological traits likely to be affected by inbreeding and selection following colonization and laboratory rearing were examined. Sperm length, and accessory gland and testes size were compared in male progeny from field-collected females and laboratory strains of Anopheles gambiae sensu stricto colonized from one to over 25 years ago. These traits were also compared in the parental and sequentially derived, genetically modified strains produced using a two-phase genetic transformation system. Finally, genetic crosses were performed between strains in order to distinguish the effects of inbreeding and selection on reproductive traits. RESULTS: Sperm length was found to steadily decrease with the age of mosquito colonies but was recovered in refreshed strains and crosses between inbred strains therefore incriminating inbreeding costs. In contrast, testes size progressively increased with colony age, whilst accessory gland size quickly decreased in males from colonies of all ages. The lack of heterosis in response to crossing and strain refreshing in the latter two reproductive traits suggests selection for insectary conditions. CONCLUSIONS: These results show that inbreeding and selection differentially affect reproductive traits in laboratory strains overtime and that heterotic 'supermales' could be used to rescue some male reproductive characteristics. Further experiments are needed to establish the exact relationship between sperm length, accessory gland and testes size, and male reproductive success in the laboratory and field settings.

A new species of phlebotomine, Trichophoromyia adelsonsouzai (Diptera: Psychodidae) of Brazilian Amazonia.

The phlebotomine sandfly Trichophoromyia adelsonsouzai sp. nov. is described and illustrated based on the male and female morphological characteristics of specimens collected at Km 27 of the Trans-Amazonian Highway, municipality of Vitória do Xingu, state of Pará, Brazilian Amazonia. This is an area subject to the direct influence of Belo Monte hydroelectric system. With the description of this new species the number of Trichophoromyia sandflies recorded in Brazil is increased to 20.

Degree of associations among vectors of the genus Culicoides (Diptera: Ceratopogonidae) and host bird species with respect to haemosporidian parasites in NE Bulgaria.

The occurrence of haemosporidians in biting midges of the genus Culicoides is examined in North-East Bulgaria in order to reveal their potential role for parasite transmission. A PCR-based technique amplifying part of the mitochondrial cytochrome b gene of the parasite is applied on naturally infected biting midges. Totally, 640 parous individuals of four species and 95 blood-fed individuals of six species of Culicoides are examined for the presence of DNA of haemosporidians. Haemosporidian genetic lineages are identified in individuals of three insect species: Culicoides alazanicus (12 lineages, nine lineages of Haemoproteus and three lineages of Plasmodium), Culicoides festivipennis and Culicoides circumscriptus (with two and one lineages of Haemoproteus, respectively). Two genetic lineages of Haemoproteus are recorded in more than one vector species. These results demonstrate variations in the specificity of Haemoproteus genetic lineages to their potential vectors, since some lineages are recorded in a single vector species and others occur in two or more vector species.