Ecological Distribution and CQ11 Genetic Structure of Culex pipiens Complex (Diptera: Culicidae) in Italy

Mosquitoes in the Culex pipiens complex are considered to be involved in the transmission of a range of pathogens, including West Nile virus (WNV). Although its taxonomic status is still debated, the complex includes species, both globally distributed or with a more limited distribution, morphologically similar and characterised by different physiological and behavioural traits, which affect their ability as vectors. In many European countries, Cx. pipiens and its sibling species Culex torrentium occur in sympatry, exhibiting similar bionomic and morphological characters, but only Cx. pipiens appears to play a vector role in WNV transmission. This species consists of two biotypes, pipiens and molestus, which can interbreed when in sympatry, and their hybrids can act as WNV-bridge vectors, due to intermediate ecological features. Considering the yearly WNV outbreaks since 2008 and given the morphological difficulties in recognising species and biotypes, our aim was to molecularly identify and characterised Cx. pipiens and Cx. torrentium in Italy, using recently developed molecular assays. Culex torrentium was not detected; as in other European countries, the pipiens and molestus biotypes were widely found in sympatry with hybrids in most environments. The UPGMA cluster analysis applied to CQ11 genotypic frequencies mainly revealed two groups of Cx. pipiens populations that differed in ecological features. The high propensity of the molestus biotype to exist in hypogean environments, where the habitat’s physical characteristics hinder and preclude the gene flow, was shown. These results confirmed the CQ11 assay as a reliable diagnostic method, consistent with the ecological and physiological aspects of the populations analysed. Since the assessment of the actual role of three biotypes in the WNV circulation remains a crucial point to be elucidated, this extensive molecular screening of Cx. pipiens populations can provide new insights into the ecology of the species and may give useful indications to plan and implement WNV surveillance activities in Italy.     

Testing of UK Populations of Culex pipiens L. for Schmallenberg Virus Vector Competence and Their Colonization

Background

Schmallenberg virus (SBV), an arboviral pathogen of ruminants, emerged in northern Europe during 2011 and has subsequently spread across a vast geographic area. While Culicoides biting midges (Diptera: Ceratopogonidae) have been identified as a biological transmission agent of SBV, the role of mosquitoes (Diptera: Culicidae) as potential vectors has not been defined beyond small-scale field collections in affected areas. Culex pipiens L. are one of the most widespread mosquitoes in northern Europe; they are present on farms across the region and have previously been implicated as vectors of several other arboviruses. We assessed the ability of three colony lines of Cx. pipiens, originating from geographically diverse field populations, to become fully infected by SBV using semi-quantitative real-time RT-PCR (sqPCR).

Findings

Two colony lines of Cx. pipiens were created in the UK (‘Brookwood’ and ‘Caldbeck’) from field collections of larvae and pupae and characterised using genetic markers. A third strain of Cx. pipiens from CVI Wageningen, The Netherlands, was also screened during experiments. Intrathoracic inoculation of the Brookwood line resulted in infections after 14 days that were characterised by high levels of RNA throughout individuals, but which demonstrated indirect evidence of salivary gland barriers. Feeding of 322 individuals across the three colony lines on a membrane based infection system resulted in no evidence of full dissemination of SBV, although infections did occur in a small proportion of Cx. pipiens from each line.

Conclusions/Significance

This study established two novel lines of Cx. pipiens mosquitoes of UK origin in the laboratory and subsequently tested their competence for SBV. Schmallenberg virus replication and dissemination was restricted, demonstrating that Cx. pipiens is unlikely to be an epidemiologically important vector of the virus in northern Europe.     

Divergent host preferences of above‐ and below‐ground Culex pipiens mosquitoes and their hybrid offspring

Culex pipiens form pipiens and Cx. pipiens form molestus (Diptera: Culicidae) belong to a cosmopolitan taxonomic group known as the Pipiens Assemblage. Hybridization between these forms is thought to contribute to human transmission of West Nile virus (WNV) in North America. Complementary choice and no‐choice landing assays were developed to examine host acceptance by North American Cx. pipiens in the laboratory. Populations collected from above‐ and below‐ground sites in suburban Chicago were identified as forms pipiens and molestus using a polymerase chain reaction‐based assay. Avian and human host acceptance was then quantified for the two populations, as well as for their hybrid and backcross offspring. No‐choice tests were used to demonstrate that both the pipiens and molestus forms were capable of feeding on human and avian hosts. Choice tests were used to demonstrate that form pipiens females were strongly avian‐seeking; an individual's probability of accepting the chick host was 85%. Form molestus females were more likely to accept the human host (87%). Rates of host acceptance by F₁ and backcross progeny were intermediate to those of their parents. The results suggest that host preferences in Cx. pipiens are genetically determined, and that ongoing hybridization between above‐ and below‐ground populations is an important contributor to epizootic transmission of WNV in North America.     

Bacteria and yeasts associated with Sirex noctilio

Five genera of bacteria and yeasts commonly occur in the oviposition drills and larval galleries of Sirex noctilio. In exceptional circumstances, some of these microorganisms may be pathogenic while the yeast, Saccharomyces sp., is involved in the production of attractants of the rhyssine parasitoids Rhyssa and Megarhyssa spp. A possible role in larval nutrition is suggested but these microorganisms have no role in the phytotoxic effects of Sirex attack.     

Evaluation of the effectiveness of the California mosquito-borne virus surveillance & response plan, 2009–2018

Local vector control and public health agencies in California use the California Mosquito-Borne Virus Surveillance and Response Plan to monitor and evaluate West Nile virus (WNV) activity and guide responses to reduce the burden of WNV disease. All available data from environmental surveillance, such as the abundance and WNV infection rates in Culex tarsalis and the Culex pipiens complex mosquitoes, the numbers of dead birds, seroconversions in sentinel chickens, and ambient air temperatures, are fed into a formula to estimate the risk level and associated risk of human infections. In many other areas of the US, the vector index, based only on vector mosquito abundance and infection rates, is used by vector control programs to estimate the risk of human WNV transmission. We built models to determine the association between risk level and the number of reported symptomatic human disease cases with onset in the following three weeks to identify the essential components of the risk level and to compare California’s risk estimates to vector index. Risk level calculations based on Cx. tarsalis and Cx. pipiens complex levels were significantly associated with increased human risk, particularly when accounting for vector control area and population, and were better predictors than using vector index. Including all potential environmental components created an effective tool to estimate the risk of WNV transmission to humans in California.     

Functional Analysis of Insect Molting Fluid Proteins on the Protection and Regulation of Ecdysis

Molting fluid accumulates between the old and new cuticles during periodical ecdysis in Ecdysozoa. Natural defects in insect ecdysis are frequently associated with melanization (an immunity response) occurring primarily in molting fluids, suggesting that molting fluid may impact immunity as well as affect ecdysis. To address this hypothesis, proteomic analysis of molting fluids from Bombyx mori during three different types of ecdysis was performed. Many proteins were newly identified, including immunity-related proteins, in each molting fluid. Molting fluids inhibited the growth of bacteria in vitro. The entomopathogenic fungi Beauveria bassiana, which can escape immune responses in feeding larvae, is quickly recognized by larvae during ecdysis, followed by melanization in molting fluid and old cuticle. Fungal conidia germination was delayed, and no hyphae were detected in the hemocoels of pharate instar insects. Molting fluids protect the delicate pharate instar insects with extremely thin cuticles against microorganisms. To explore the function of molting fluids in ecdysis regulation, based on protein similarity, 32 genes were selected for analysis in ecdysis regulation through RNAi in Tribolium castaneum, a model commonly used to study integument development because RNAi is difficult to achieve in B. mori. We identified 24 molting proteins that affected ecdysis after knockdown, with different physiological functions, including old cuticle protein recycling, molting fluid pressure balance, detoxification, and signal detection and transfer of molting fluids. We report that insects secrete molting fluid for protection and regulation of ecdysis, which indicates a way to develop new pesticides through interrupting insect ecdysis in the future.     

Intestinal microbiota of gibel carp (Carassius auratus gibelio) and its origin as revealed by 454 pyrosequencing

The intestinal microbiota has received increasing attention, as it influences growth, feed conversion, epithelial development, immunity as well as the intrusion of pathogenic microorganisms in the intestinal tract. In this study, pyrosequencing was used to explore the bacterial community of the intestine in gibel carp (Carassius auratus gibelio), and the origin of these microorganisms. The results disclosed great bacterial diversities in the carp intestines and cultured environments. The gibel carp harbored characteristic intestinal microbiota, where Proteobacteria were predominant, followed by Firmicutes. The analysis on the 10 most abundant bacterial operational taxonomic units (OTUs) revealed a majority of Firmicutes in the intestinal content (by decreasing order: Veilonella sp., Lachnospiraceae, Lactobacillales, Streptococcus sp., and Lactobacillus sp.). The second most abundant OTU was Rothia sp. (Actinobacteria). The most likely potential probiotics (Lactobacillus sp., and Bacillus sp.) and opportunists (Aeromonas sp., and Acinetobacter sp.) were not much abundant. Bacterial community comparisons showed that the intestinal community was closely related to that of the sediment, indicating the importance of sediment as source of gut bacteria in gibel carp. However, 37.95 % of the OTUs detected in feed were retrieved in the intestine, suggesting that food may influence markedly the microbiota of gibel carp, and therefore may be exploited for oral administration of probiotics.     

Chromosome-level assembly of Culex pipiens molestus and improved reference genome of Culex pipiens pallens (Culicidae,Diptera)

Culex pipiens molestus and Culex pipiens pallens are two distinct bioforms in the Culex pipiens complex that are important vectors of several pathogens and are widely distributed around the world. In the current study, we present a high-quality chromosome-level genome of Cx. pipiens f. molestus and describe the genetic characteristics of this genome. The assembly genome was 559.749 Mb with contig and scaffold N50 values of 200.952 Mb and 0.370 Mb, and more than 94.78% of the assembled bases were located on 3 chromosomes. A total of 19,399 protein-coding genes were predicted. Many gene families were expanded in the genome of Cx. pipiens f. molestus, particularly those of the chemosensory protein (CSP) and gustatory receptor (GR) gene families. In addition, utilizing Hi-C data, we improved the previously assembled draft genome of Cx. pipiens f. pallens, with scaffold N50 of 186.195 Mb and contig N50 of 0.749 Mb, and more than 97.02% of the assembled bases were located on three chromosomes. This reference genome provides a foundation for genome-based investigations of the unique ecological and evolutionary characteristics of Cx. pipiens f. molestus, and the findings in this study will help to elucidate the mechanisms involved in species divergence in the Culex pipiens complex.     

Impacts of fungal entomopathogens on survival and immune responses of Aedes albopictus and Culex pipiens mosquitoes in the context of native Wolbachia infections

Microbial control of mosquitoes via the use of symbiotic or pathogenic microbes, such as Wolbachia and entomopathogenic fungi, are promising alternatives to synthetic insecticides to tackle the rapid increase in insecticide resistance and vector-borne disease outbreaks. This study evaluated the susceptibility and host responses of two important mosquito vectors, Ae. albopictus and Cx. pipiens, that naturally carry Wolbachia, to infections by entomopathogenic fungi. Our study indicated that while Wolbachia presence did not provide a protective advantage against entomopathogenic fungal infection, it nevertheless influenced the bacterial / fungal load and the expression of select anti-microbial effectors and phenoloxidase cascade genes in mosquitoes. Furthermore, although host responses from Ae. albopictus and Cx. pipiens were mostly similar, we observed contrasting phenotypes with regards to susceptibility and immune responses to fungal entomopathogenic infection in these two mosquitoes. This study provides new insights into the intricate multipartite interaction between the mosquito host, its native symbiont and pathogenic microbes that might be employed to control mosquito populations.     

Mosquito population structure,pathogen surveillance and insecticide resistance monitoring in urban regions of Crete,Greece

BackgroundIn Greece vector borne diseases (VBD) and foremost West Nile virus (WNV) pose an important threat to public health and the tourist industry, the primary sector of contribution to the national economy. The island of Crete, is one of Greece’s major tourist destinations receiving annually over 5 million tourists making regional VBD control both a public health and economic priority.MethodologyUnder the auspices of the Region of Crete, a systematic integrative surveillance network targeting mosquitoes and associated pathogens was established in Crete for the years 2018–2020. Using conventional and molecular diagnostic tools we investigated the mosquito species composition and population dynamics, pathogen infection occurrences in vector populations and in sentinel chickens, and the insecticide resistance status of the major vector species.Principal findingsImportant disease vectors were recorded across the island including Culex pipiens, Aedes albopictus, and Anopheles superpictus. Over 75% of the sampled specimens were collected in the western prefectures potentially attributed to the local precipitation patterns, with Cx. pipiens being the most dominant species. Although no pathogens (flaviviruses) were detected in the analysed mosquito specimens, chicken blood serum analyses recorded a 1.7% WNV antibody detection rate in the 2018 samples. Notably detection of the first WNV positive chicken preceded human WNV occurrence in the same region by approximately two weeks. The chitin synthase mutation I1043F (associated with high diflubenzuron resistance) was recorded at an 8% allelic frequency in Lasithi prefecture Cx. pipiens mosquitoes (sampled in 2020) for the first time in Greece. Markedly, Cx. pipiens populations in all four prefectures were found harboring the kdr mutations L1014F/C/S (associated with pyrethroid resistance) at a close to fixation rate, with mutation L1014C being the most commonly found allele (≥74% representation). Voltage gated sodium channel analyses in Ae. albopictus revealed the presence of the kdr mutations F1534C and I1532T (associated with putative mild pyrethroid resistance phenotypes) yet absence of V1016G. Allele F1534C was recorded in all prefectures (at an allelic frequency range of 25–46.6%) while I1532T was detected in populations from Chania, Rethymnon and Heraklion (at frequencies below 7.1%). Finally, no kdr mutations were detected in the Anopheles specimens included in the analyses.Conclusions/SignificanceThe findings of our study are of major concern for VBD control in Crete, highlighting (i) the necessity for establishing seasonal integrated entomological/pathogen surveillance programs, supporting the design of targeted vector control responses and; ii) the need for establishing appropriate insecticide resistance management programs ensuring the efficacy and sustainable use of DFB and pyrethroid based products in vector control.     

Culex quinquefasciatus larvae development arrested when fed on Neochloris aquatica

Culex quinquefasciatus is a cosmopolitan species widely distributed in the tropical and subtropical areas of the world. Due to its long history of close association with humans, the transmission of arboviruses and parasites have an important role in veterinary and public health. Adult females feed mainly on birds although they can also feed on humans and other mammals. On the other hand, larvae are able to feed on a great diversity of microorganisms, including microalgae, present in natural or artificial breeding sites with a high organic load. These two particularities, mentioned above, are some of the reasons why this mosquito is so successful in the environment. In this work, we report the identification of a microalga found during field sampling in artificial breeding sites, in a group of discarded tires with accumulated rainwater. Surprisingly, only one of them had a bright green culture without mosquito larvae while the other surrounding tires contained a large number of mosquito larvae. We isolated and identified this microorganism as Neochloris aquatica, and it was evaluated as a potential biological control agent against Cx. quinquefasciatus. The oviposition site preference in the presence of the alga by gravid females, and the effects on larval development were analyzed. Additionally, microalga effect on Cx. quinquefasciatus wild type, naturally infected with the endosymbiotic bacterium Wolbachia (w⁺) and Wolbachia free (w⁻) laboratory lines was explored. According to our results, even though it is chosen by gravid females to lay their eggs, the microalga had a negative effect on the development of larvae from both populations. Additionally, when the larvae were fed with a culture of alga supplemented with balanced fish food used as control diet, they were not able to reverse its effect, and were unable to complete development until adulthood. Here, N. aquatica is described as a biological agent, and as a potential source of bioactive compounds for the control of mosquito populations important in veterinary and human health.     

Knickkopf and retroactive proteins are required for formation of laminar serosal procuticle during embryonic development of Tribolium castaneum

Chitin, a homopolymer of β-1-4-linked N-acetylglucosamine synthesized by chitin synthase A (Chs-A), is organized in the procuticle of the postembryonic cuticle or exoskeleton, which is composed of laminae stacked parallel to the cell surface to give stability and integrity to the underlying insect epidermal and other tissues. Our previous work has revealed an important role for two proteins from Tribolium castaneum named Knickkopf (TcKnk) and Retroactive (TcRtv) in postembryonic cuticular chitin maintenance. TcKnk and TcRtv were shown to be required for protection and organization of newly synthesized procuticular chitin. To study the functions of TcKnk and TcRtv in serosal and larval cuticles produced during embryogenesis in T. castaneum, dsRNAs specific for these two genes were injected into two week-old adult females. The effects of dsRNA treatment on ovarial integrity, oviposition, egg hatching and adult survival were determined. Insects treated with dsRNA for chitin synthase-A (TcChs-A) and tryptophan oxygenase (TcVer) were used as positive and negative controls for these experiments, respectively. Like TcChs-A RNAi, injection of dsRNA for TcKnk or TcRtv into adult females exhibited no adult lethality and oviposition was normal. However, a vast majority of the embryos did not hatch. The remaining (∼10%) of the embryos hatched into first instar larvae that died without molting to the second instar. Chitin content analysis following TcKnk and TcRtv parental RNAi revealed approximately 50% reduction in chitin content of eggs in comparison with control TcVer RNAi, whereas TcChs-A dsRNA-treatment led to >90% loss of chitin. Furthermore, transmission electron microscopic (TEM) analysis of serosal cuticle from TcChs-A, TcKnk and TcRtv dsRNA-treated insects revealed a complete absence of laminar organization of serosal (and larval) procuticle in comparison with TcVer dsRNA-treated controls, which exhibited normal laminar organization of procuticular chitin. The results of this study demonstrate that in addition to their essential roles in maintenance and organization of chitin in epidermal cuticle in larval and later stages of insect development, TcKnk and TcRtv also are required for egg hatch, chitin maintenance and laminar organization of both serosal and larval cuticle during embryonic development of T. castaneum.     

我国尖音库蚊复合组蚊虫的杂交及其与Wolbachia感染的关系

为了了解我国尖音库蚊复合组蚊虫间杂交卵的不孵化现象和明确该现象与共生微生物Wolbachia感染的关系,对该复合组实验室种群4个亚种进行了笼内杂交和抗生素处理后的杂交。试验表明: 在复合组蚊虫中骚扰库蚊Culex pipiens molestus与淡色库蚊Cx. Pipiens pallens、致倦库蚊Cx. Ipiens quinquefasciatus与尖音库蚊Cx. Pipiens pipiens之间存在有单向胞质不融合现象,骚扰库蚊的雄虫与尖音库蚊、致倦库蚊和淡色库蚊的雌虫杂交卵的孵化率分别为0.06%、0.46%和0.19%;该胞质不融合现象可以通过抗生素处理而消除,处理后骚扰库蚊雄虫与其余3个亚种雌虫F₃杂交卵的孵化率均有提高,分别为89.49%（t=3.90×10^-28＜t_0.01=2.704）、23.39%（t=9.15×10^-7＜t_0.01=2.660_）和22.27%（t=5.08×10^-4＜t_0.01=2.750）,并可因抗生素处理而产生新的不融合类型。     

Microbial Community Structure at Different Fermentation Stages of Kutajarista, a Herbal Formulation

Kutajarista is an Ayurvedic fermented herbal formulation prescribed for gastrointestinal disorders. This herbal formulation undergoes a gradual fermentative process and takes around 2 months for production. In this study, microbial composition at initial stages of fermentation of Kutajarista was assessed by culture independent 16S rRNA gene clone library approach. Physicochemical changes were also compared at these stages of fermentation. High performance liquid chromatography–mass spectrometry analysis showed that Gallic acid, Ellagic acid, and its derivatives were the major chemical constituents recovered in this process. At 0 day of fermentation, Lactobacillus sp., Acinetobacter sp., Alcaligenes sp., and Methylobacterium sp. were recovered, but were not detected at 8 day of fermentation. Initially, microbial diversity increased after 8 days of fermentation with 11 operational taxonomic units (OTUs), which further decreased to 3 OTUs at 30 day of fermentation. Aeromonas sp., Pseudomonas sp., and Klebsiella sp. dominated till 30 day of fermentation. Predominance of γ- Proteobacteria and presence of gallolyl derivatives at the saturation stage of fermentation implies tannin degrading potential of these microbes. This is the first study to highlight the microbial role in an Ayurvedic herbal product fermentation.     

Severe developmental timing defects in the prothoracicotropic hormone (PTTH)-deficient silkworm,Bombyx mori

The insect neuropeptide prothoracicotropic hormone (PTTH) triggers the biosynthesis and release of the molting hormone ecdysone in the prothoracic gland (PG), thereby controlling the timing of molting and metamorphosis. Despite the well-documented physiological role of PTTH and its signaling pathway in the PG, it is not clear whether PTTH is an essential hormone for ecdysone biosynthesis and development. To address this question, we established and characterized a PTTH knockout line in the silkworm, Bombyx mori. We found that PTTH knockouts showed a severe developmental delay in both the larval and pupal stages. Larval phenotypes of PTTH knockouts can be classified into three major classes: (i) developmental arrest during the second larval instar, (ii) precocious metamorphosis after the fourth larval instar (one instar earlier in comparison to the control strain), and (iii) metamorphosis to normal-sized pupae after completing the five larval instar stages. In PTTH knockout larvae, peak levels of ecdysone titers in the hemolymph were dramatically reduced and the timing of peaks was delayed, suggesting that protracted larval development is a result of the reduced and delayed synthesis of ecdysone in the PG. Despite these defects, low basal levels of ecdysone were maintained in PTTH knockout larvae, suggesting that the primary role of PTTH is to upregulate ecdysone biosynthesis in the PG during molting stages, and low basal levels of ecdysone can be maintained in the absence of PTTH. We also found that mRNA levels of genes involved in ecdysone biosynthesis and ecdysteroid signaling pathways were significantly reduced in PTTH knockouts. Our results provide genetic evidence that PTTH is not essential for development, but is required to coordinate growth and developmental timing.     

Sublethal effects of chronic exposure to chlorpyrifos or imidacloprid insecticides or their binary mixtures on Culex pipiens mosquitoes

Mosquitoes represent one of the most significant threats to human and veterinary health throughout the world. Consequently, improving strategies for the control of mosquitoes is essential. In the present study, juvenile Culex pipiens (Diptera: Culicidae), the common house mosquito, are chronically exposed to sublethal concentrations of chlorpyrifos (20% of LC₅₀) and imidacloprid (5% of LC₅₀), both separately and as a mixture. Developmental time, the emergence rate of adults and the expression of five selected genes involved in detoxification and resistance to pesticides are assessed. To assess the effects on oviposition choice, gravid females are forced to oviposit into cups containing water with added chlorpyrifos, imidacloprid or a mixture of both. The time required for the development of second‐ and third‐instar larvae is observed to differ significantly between treatments. Adults of C. pipiens fail to emerge from larvae hatched in both imidacloprid and the binary mixture. The expression of the four quantified detoxification genes differs significantly in third‐larval instars exposed to chlorpyrifos and/or imidacloprid compared with controls. Gravid females also fail to lay eggs on water to which either of the insecticides or the binary mixture is added, although they do lay eggs in cups containing water only. Chronic exposure to sublethal concentrations of chlorpyrifos or imidacloprid has significant adverse effects on development and thus the reproductive fitness of C. pipiens and, accordingly, could be used in the population control of these mosquitoes.     

Sampling Design Influences the Observed Dominance of Culex tritaeniorhynchus: Considerations for Future Studies of Japanese Encephalitis Virus Transmission

Mosquito sampling during Japanese encephalitis virus (JEV)-associated studies, particularly in India, has usually been conducted via aspirators or light traps to catch mosquitoes around cattle, which are dead-end hosts for JEV. High numbers of Culex tritaeniorhynchus, relative to other species, have often been caught during these studies. Less frequently, studies have involved sampling outdoor resting mosquitoes. We aimed to compare the relative abundance of mosquito species between these two previously used mosquito sampling methods. From September to December 2013 entomological surveys were undertaken in eight villages in a Japanese encephalitis (JE) endemic area of Bangladesh. Light traps were used to collect active mosquitoes in households, and resting boxes and a Bina Pani Das hop cage were used near oviposition sites to collect resting mosquitoes. Numbers of humans and domestic animals present in households where light traps were set were recorded. In five villages Cx. tritaeniorhynchus was more likely to be selected from light trap samples near hosts than resting collection samples near oviposition sites, according to log odds ratio tests. The opposite was true for Cx. pseudovishnui and Armigeres subalbatus, which can also transmit JEV. Culex tritaeniorhynchus constituted 59% of the mosquitoes sampled from households with cattle, 28% from households without cattle and 17% in resting collections. In contrast Cx. pseudovishnui constituted 5.4% of the sample from households with cattle, 16% from households with no cattle and 27% from resting collections, while Ar. subalbatus constituted 0.15%, 0.38%, and 8.4% of these samples respectively. These observations may be due to differences in timing of biting activity, host preference and host-seeking strategy rather than differences in population density. We suggest that future studies aiming to implicate vector species in transmission of JEV should consider focusing catches around hosts able to transmit JEV.     

Proposal to assign Aeromonas diversa sp. nov. as a novel species designation for Aeromonas group 501

The Aeromonas group 501, also named Aeromonas sp. HG13, is taxonomically close to A. schubertii. Results obtained in previous studies, including DNA–DNA hybridization and DNA fingerprinting, suggest that Aeromonas group 501 could constitute a different Aeromonas species. In this work we have performed a polyphasic study with the two strains comprising the Aeromonas sp. HG13 in order to propose a formal species name. They could be differentiated from A. schubertii by the indole and lysine decarboxylase tests and the utilization of l-lactate. Phenotypically, both strains were also easily separated from the other Aeromonas species. Sequence analysis of the 16S rRNA gene showed high sequence similarities (>97%) between Aeromonas group 501 and all Aeromonas species. Nevertheless, sequence divergences of cpn60, dnaJ, gyrB and rpoD genes were higher than the intraspecific threshold values established for each gene (3.5%, 3.3%, 2.3% and 2.6%, respectively), while sequence divergences between strains CDC 2478-85^T and CDC 2555-87 were low (0.6–1.1%). The DNA G+C content of the type strain was 62.2 mol%. Phenotypic and genotypic evidence strongly suggests that the Aeromonas group 501 is a novel species of the genus Aeromonas, for which the name Aeromonas diversa sp. nov. is proposed. The type strain is CDC 2478-85^T (=CECT 4254^T=ATCC 43946^T=LMG 17321^T).     

Regulation of development of the grain beetle Tenebrio molitor by neuropeptides

Summary

Two neuropeptides (NPs), hydra head activator (HHA), and the luteinizing hormone releasing hormone (LHRH), were used to study their effects on the development of the larvae of the grain beetle Tenebrio molitor (mealworms). The larvae received the NPs in the course of development at the beginning or at the end of instar. The LHRH given at the beginning of the instar stimulated molting and pupation in larvae; it did not affect molting in the late period of the instar. The C-end fragment of LHRH_(9–10) produced fewer molts and increased the duration of the instar period. The effect of this fragment was weak when it was injected at the initial period of the instar; it was considerably stronger in the end of the instar. It inhibited development when administered during the last instar. On the contrary, the N-end fragment of the LHRH_(1–2) stimulated growth and development of larvae. The action of these NPs can be connected to the function of the hormones of metamorphosis. The HHA administrated to the larvae at different days of the instar stimulated molting but in the last instar it delayed pupation. The C-end of the HHA_(7–11) stimulated molting when injected during the whole period of the instar. The other C-end fragments, the HHA_(8–11) and HHA_(9–11) had inhibitory effects on the molting. The injection of the N-end fragment of the HHA_(1–5) had no effect. The action of HHA and its fragments did not correlate with the function of the hormones of metamorphosis.