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

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          Abstract

          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.

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          MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms.

          The Molecular Evolutionary Genetics Analysis (Mega) software implements many analytical methods and tools for phylogenomics and phylomedicine. Here, we report a transformation of Mega to enable cross-platform use on Microsoft Windows and Linux operating systems. Mega X does not require virtualization or emulation software and provides a uniform user experience across platforms. Mega X has additionally been upgraded to use multiple computing cores for many molecular evolutionary analyses. Mega X is available in two interfaces (graphical and command line) and can be downloaded from www.megasoftware.net free of charge.
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            Mitochondrial haplotypes and phylogeography of Phlebotomus vectors of Leishmania major.

            Haplotypes of eight phlebotomine species were characterized by cycle sequencing a mitochondrial (mt) DNA fragment (cytochrome b to NADH1) amplified from single sandflies by PCR. Phlebotomus (Phlebotomus) papatasi displayed little variation throughout its large geographical range. We conclude that this vector of Leishmania major suffered a population bottleneck late in the Pleistocene and then radiated out from the eastern Mediterranean subregion. There was no support for a recent domestic lineage of P. papatasi. The mtDNA molecular clock in phlebotomines (subgenera Phlebotomus and Larroussius) was calibrated by reference to palaeogeographical events in Africa and the Mediterranean subregion. It fitted a pairwise nucleotide sequence divergence rate of 1.0-2.5% per million years. Co-evolution of L. major, its Phlebotomus vectors and mammalian reservoirs is discussed.
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              The phlebotomine sand flies (Diptera, Psychodidae) of the oriental region

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                Author and article information

                Contributors
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Funding acquisitionRole: MethodologyRole: Project administration
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Formal analysisRole: ResourcesRole: Writing – review & editing
                Role: Formal analysisRole: ValidationRole: Writing – review & editing
                Role: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS One
                plos
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                25 February 2022
                2022
                : 17
                : 2
                : e0263268
                Affiliations
                [1 ] Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
                [2 ] Vector Biology and Vector Borne Diseases Research Unit, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
                [3 ] Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
                [4 ] Medical Science Research Equipment Center, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
                [5 ] Faculté de Pharmacie, Université de Reims Champagne-Ardenne, SFR Cap Santé, EA7510 ESCAPE–USC ANSES VECPAR, Reims, France
                Universidade Federal da Bahia, BRAZIL
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                https://orcid.org/0000-0002-5338-6213
                https://orcid.org/0000-0001-9305-1824
                https://orcid.org/0000-0001-6995-0088
                https://orcid.org/0000-0002-9248-1678
                Article
                PONE-D-21-33676
                10.1371/journal.pone.0263268
                8880748
                35213563
                4f93f051-7483-40b1-8fdc-5e6a55a225bf
                © 2022 Jariyapan et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 21 October 2021
                : 14 January 2022
                Page count
                Figures: 8, Tables: 2, Pages: 17
                Funding
                Funded by: The Program Management Unit for Human Resources & Institutional Development, Research and Innovation - Chulalongkorn University
                Award ID: B16F630071
                Award Recipient :
                Funded by: The Thailand Science Research and Innovation (TSRI) Fund
                Award ID: CU_FRB640001_01_30_1
                Award Recipient :
                Funded by: The Faculty of Medicine Research Fund, Chiang Mai University
                Award ID: 021-2563
                Award Recipient :
                This work was supported by the Program Management Unit for Human Resources & Institutional Development, Research and Innovation - Chulalongkorn University [grant number B16F630071] received by NJ; the Thailand Science Research and Innovation (TSRI) Fund [grant number CU_FRB640001_01_30_1] received by NJ, https://www.chula.ac.th/en/, and https://www.tsri.or.th/; and the Faculty of Medicine, Chiang Mai University [grant number 021-2563] received by PT, https://www.med.cmu.ac.th. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Physiology
                Reproductive Physiology
                Eggs
                Medicine and Health Sciences
                Medical Conditions
                Infectious Diseases
                Disease Vectors
                Insect Vectors
                Sand Flies
                Biology and Life Sciences
                Species Interactions
                Disease Vectors
                Insect Vectors
                Sand Flies
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Germ Cells
                OVA
                Biology and Life Sciences
                Zoology
                Entomology
                Insects
                Diptera
                Phlebotomus
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Invertebrates
                Arthropoda
                Insects
                Diptera
                Phlebotomus
                Biology and Life Sciences
                Zoology
                Animals
                Invertebrates
                Arthropoda
                Insects
                Diptera
                Phlebotomus
                Research and Analysis Methods
                Microscopy
                Electron Microscopy
                Scanning Electron Microscopy
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Biology and Life Sciences
                Anatomy
                Digestive System
                Teeth
                Medicine and Health Sciences
                Anatomy
                Digestive System
                Teeth
                Biology and Life Sciences
                Anatomy
                Head
                Jaw
                Teeth
                Medicine and Health Sciences
                Anatomy
                Head
                Jaw
                Teeth
                Physical Sciences
                Materials Science
                Materials
                Pigments
                Custom metadata
                All nucleotide sequences are available from the GenBank database (accession numbers OK398062-OK398083).

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