Daiki Mizushima

Background: In tropical to subtropical regions, centipede bites may prompt medical attention, with manifestations largely reflecting venom-related discomfort, although infections, including rare fatal necrotizing soft tissue infection (NSTI), have been reported. However, no reports are available on the commensal bacteria on centipede forcipules. Objectives: This study aimed to investigate bacterial species residing on and in centipede forcipules and their potential role in post-bite infections. Methods: Nine Scolopendra mutilans , three Scolopendra japonica , and two Bothropolys rugosus were collected from three regions in Japan. The bacterial composition of their forcipules was analyzed using 16S ribosomal ribonucleic acid gene sequencing and microbiome analysis. Findings: A diverse bacterial community was observed on the centipede forcipules. Among the NSTI-associated genera examined ( Escherichia, Staphylococcus, and Streptococcus ), only Staphylococcus was identified as a minor population. Conclusion: This study provides the first evidence that some bacteria found on centipede forcipules have been previously isolated from centipede bite infections. The risk of infection from bacteria on centipede forcipules during a centipede bite appears low. However, the presence of diverse bacterial species emphasizes the importance of thoroughly cleaning centipede bite wounds to prevent secondary infection.

Anopheles mosquitoes spread malaria, which is caused by Plasmodium parasite infection. In nature, both male and female mosquitoes typically ingest sugars from floral nectar; however, few studies have investigated the effects of sugars as an insecticide for mosquitoes. During our previous work, the lifespan of An. stephensi was shortened by the feeding of high concentrations of lactose. The mechanism by which lactose can shorten the lifespan of mosquitoes is still unidentified. The present study aims to evaluate the potential of lactose as an insecticide against mosquitoes. A sublethal concentration of lactose decreased the lifespan of An. stephensi but did not affect their blood-feeding ratio or the number of eggs they laid. Female Anopheles mosquitoes refused to ingest lactose-containing sugars when a lactose-free sugar is available; however, male mosquitoes ingest them. Both sexes excreted lactose from their bodies after feeding, while other sugars were completely absorbed and digested. An ingestion volume assay using food dye revealed that lactose reduces the volume of the solution in the mosquito body. Female mosquitoes forced to ingest lactose for 3 wk showed a slight change in the composition of their midgut microbiota but not in their relative quantity. These findings suggest that lactose feeding causes osmotic diarrhea-like symptoms in Anopheles mosquitoes. The use of attractive toxic sugar baits with lactose as an insecticide is expected to control male Anopheles mosquitoes.

Background: Blood-sucking phlebotomine sand flies are vectors of the protozoan parasites Leishmania spp. Although the intestinal microbiota is involved in a wide range of biological and physiological processes and has the potential to alter vector competence, little is known about the factors that modify the gut microbiota composition of sand flies. As a key step toward addressing this issue, we investigated the impact of host species on the gut bacterial composition in Phlebotomus and Lutzomyia sand flies reared under the same conditions. Methods: Bacterial 16S rRNA gene amplification and Illumina MiSeq sequencing were used to characterize the overall bacterial composition of three laboratory-reared sandflies: Phlebotomus papatasi, Ph. duboscqi, and Lutzomyia longipalpis. Results: Our results showed that the larvae of the three sand fly species harbored almost the same microbes but had different relative abundances. Adult Ph. papatasi and Ph. duboscqi revealed similar microbiome compositions, which were distinct from that of adult Lu. longipalpis. Furthermore, we showed that Ph. papatasi and Ph. duboscqi are hosts for different bacterial genera. The experiment was repeated twice to improve accuracy and increase reliability of the data, and the same results were obtained even when a distinct composition of the microbiome among the same species was identified probably because of the use of different larvae food batch. Conclusions: The present study provides key insights into the role of host species in the gut microbial content of different sand fly species reared under the same conditions, which may influence their susceptibility to Leishmania infection. Graphical

A rare sugar, allose, was reported to inhibit the development of Plasmodium parasites in Anopheles mosquitoes; however, the mechanism remains unknown. The present study addressed the inhibitory mechanism of allose on the development of the Plasmodium parasite by connecting it with bacteria involvement in the midgut. In addition, further inhibitory sugars against Plasmodium infection in mosquitoes were explored. Antibiotic-treated and antibiotic-untreated Anopheles stephensi were fed fructose with or without allose. The mosquitoes were infected with luciferase-expressing Plasmodium berghei, and parasite development was evaluated by luciferase activity. Bacterial composition analysis in gut of their mosquitoes was performed with comprehensive 16S ribosomal RNA sequencing. As the result, allose inhibited the development of oocysts in mosquitoes regardless of prior antibiotic treatment. Microbiome analysis showed that the midgut bacterial composition in mosquitoes before and after blood feeding was not affected by allose. Although allose inhibited transient growth of the midgut microbiota of mosquitoes after blood feeding, neither toxic nor inhibitory effects of allose on the dominant midgut bacteria were observed. Ookinete development in the mosquito midgut was also not affected by allose feeding. Additional 15 sugars including six monosaccharides, four polyols, and five polysaccharides were tested; however, no inhibitory effect against Plasmodium development in mosquitoes was observed. These results indicated that allose inhibits parasite development in midgut stage of the mosquito independently of midgut microbiota. Although further studies are needed, our results suggest that allose may be a useful material for the vector control of malaria as a “transmission-blocking sugar.”

Sand flies are a significant public health concern in many parts of the world where they are known to transmit agents of several zoonotic diseases to humans, such as leishmaniasis. Vector control remains a key component of many anti-leishmaniasis programs and probably will remain so until an effective vaccine becomes available. The sand fly gut microbiota has recently emerged as an encouraging field for the exploration of vector-based disease control. In particular, the gut microbiome was previously reported to either enhance or inhibit parasite activity depending on the species of bacteria and, thus, has the potential to alter vector competence. Here, we describe the technological advances that are currently expanding our understanding of microbiota composition in sand flies. The acquisition and composition of microbiomes are influenced by several abiotic and biotic factors, including host immunity, genetics, and the environment. Therefore, the microbiomes of sand flies can vary substantially between individuals, life stages, species, and over geographical space, and this variation likely contributes to differences in host phenotypes, highlighting opportunities for novel vector control strategies.

Differences in the gut microbial content of Lutzomyia (Lu.) ayacuchensis, a primary vector of Andean-type cutaneous leishmaniasis in Ecuador and Peru, may influence the susceptibility of these sand flies to infection by Leishmania. As a first step toward addressing this hypothesis, a comparative analysis of bacterial and fungal compositions from Lu. ayacuchensis populations with differential susceptibilities to Leishmania was performed. Bacterial 16S rRNA gene amplification and Illumina MiSeq sequencing approaches were used to characterize the bacterial composition in wild-caught populations from the Andean areas of Ecuador and southern Peru at which the sand fly species transmit Leishmania (Leishmania) mexicana and Leishmania (Viannia) peruviana, respectively, and a population from the northern Peruvian Andes at which the transmission of Leishmania by Lu. ayacuchensis has not been reported. In the present study, 59 genera were identified, 21 of which were widely identified and comprised more than 95% of all bacteria. Of the 21 dominant bacterial genera identified in the sand flies collected, 10 genera had never been detected in field sand flies. The Ecuador and southern Peru populations each comprised individuals of particular genera, while overlap was clearly observed between microbes isolated from different sites, such as the number of soil organisms. Similarly, Corynebacterium and Micrococcus were slightly more dominant bacterial genera in the southern Peru population, while Ochrobactrum was the most frequently isolated from other populations. On the other hand, fungi were only found in the southern Peru population and dominated by the Papiliotrema genus. These results suggest that variation in the insect gut microbiota may be elucidated by the ecological diversity of sand flies in Peru and Ecuador, which may influence susceptibility to Leishmania infection. The present study provides key insights for understanding the role of the microbiota during the course of L. (L.) mexicana and L. (V.) peruviana infections in this important vector.

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mannose phosphate isomerase (mpi) gene was applied to 134 skin samples collected from patients with cutaneous leishmaniasis (CL) in Peru for identification of the infecting parasite at the species level, and the results were compared with those of cytochrome b (cyt b) gene sequencing obtained in previous studies. Although most results (121/134) including 4 hybrids of Leishmania (Viannia) braziliensis and L. (V.) peruviana corresponded to those obtained in the previous study, PCR-RFLP analyses revealed the distribution of putative hybrid strains between L. (V.) peruviana and L. (V.) lainsoni in two samples, which has never been reported. Moreover, parasite strains showing discordance between kinetoplast and nuclear genes (kDNA and nDNA), so-called mito-nuclear discordance, were identified in 11 samples. Of these, six strains had the kDNAs of L. (V.) braziliensis or L. (V.) peruviana and nDNAs of L. (V.) guyanensis, and three strains had the kDNAs of L. (V.) shawi and nDNAs of L. (V.) braziliensis. The rest were identified as mito-nuclear discordance strains having kDNAs of L. (V.) braziliensis or L. (V.) peruviana and nDNAs of L. (V.) lainsoni, and kDNAs of L. (V.) lainsoni and nDNAs of L. (V.) braziliensis. The results demonstrate that Leishmania strains in Peru are genetically more complex than previously considered. Protozoan parasites of the genus Leishmania are able to undergo genetic exchange during their growth. The previous description of hybrids in Peru and the recent discovery of unexpected genetically complex strains having characteristics of both hybrid and mito-nuclear discordance in its neighbouring country (Ecuador) with a similar eco-epidemiological situation led us to consider that the genetic structure of Leishmania strains in Peru is more complicated than previously thought. In an effort to revise the data on Leishmania strain dispersion in Peru and to search for evidence of genetic recombination, the present study was conducted. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis targeting the mannose phosphate isomerase (mpi) gene sequence was performed to identify the infecting parasite at the species level in 134 skin samples collected from patients with cutaneous leishmaniasis (CL) in Peru, and the results were compared with those of cytochrome b (cyt b) gene sequencing obtained in previous studies. Most results (121/134) including 4 hybrids between L. (V.) braziliensis and L. (V.) peruviana showed agreement between PCR-RFLP of the mpi gene and cyt b gene sequence analysis; however, 13 of 134 samples revealed the distribution of strains with hybrids and mito-nuclear discordance. The results demonstrate that genetically complex Leishmania strains are present in Peru. These findings indicate that Leishmania strain dispersion in Peru is genetically more complex than previously considered. Further prospective studies including larger samples and the isolation of parasite strains are required to update the available data.