Polymerase chain reaction and gyrA nucleotide sequence analysis of Wolbachia endosymbionts (Rickettsiales: Anaplasmataceae) in various species of Culicidae, Cimex lectularius (Hemiptera: Cimicidae) and Dirofilaria immitis (Rhabditida: Onchocercidae)

Authors

  • Jennifer R Bess Hillsborough Community College, Department of Biology, Tampa, Florida 33614, USA
  • Vincent Centonze Hillsborough Community College, Veterinary Technology Program, Plant City, Florida 33563, USA
  • Cody Rawls Hillsborough Community College, Department of Biology, Tampa, Florida 33614, USA
  • Andrew White Hillsborough Community College, Department of Biology, Tampa, Florida 33614, USA
  • David Wingfield Hillsborough Community College, Department of Biology, Tampa, Florida 33614, USA
  • John Edward Whitlock Hillsborough Community College, Department of Biology, Tampa, Florida 33614, USA

Abstract

Wolbachia Hertig and Wolbach (Rickettsiales: Anaplasmataceae) are non-culturable, bacterial endosymbionts that have been found in a broad range of arthropods and other invertebrate species. They have been implicated in human and veterinary pathologies, and may play a major role in embryonic development and evolution of host species. Given the apparent ubiquity of Wolbachia in certain animal taxa suggested by previous studies, there are still many unanswered questions about its biology. Like other obligate intracellular bacteria, they are difficult to cultivate outside of their host and often are analyzed using molecular methods. Polymerase chain reaction (PCR) assays have been developed previously for Wolbachia detection within host species, and several genes have been explored for strain typing and phylogenetic reconstruction. However, given the expansive host range and biological complexity of symbiotic relationships between Wolbachia and its host species, new methods could help accelerate the pace of Wolbachia research. As part of an overarching goal to study the distribution of Wolbachia in local mosquitoes and in the heartworm, Dirofilaria immitis (Leidy) (Rhabditida: Onchocercidae), we aimed to develop cost-effective methods that can be used in strain identification and analysis. We developed a novel PCR assay targeting the gyrA gene of Wolbachia and explored various methods of sample preparation. Presumptive Wolbachia were detected in mosquito specimens from several genera, as well as from D. immitis samples obtained from canine necropsy. DNA sequence analysis of the PCR products confirmed the identity of Wolbachia and revealed variability within some regions of the gyrA gene that correspond to host species. Consequently, this gene could be useful for future phylogenetic and population studies.

Resumen

Wolbachia Hertig y Wolbach (Rickettsiales: Anaplasmataceae) son endosimbiontes bacterianos no cultivables que se han encontrado en una amplia gama de artrópodos y otras especies de invertebrados. Se han implicado en patologías humanas y veterinarias y pueden jugar un papel importante en el desarrollo embrionario y la evolución de las especies hospederas. Dada la aparente ubicuidad de Wolbachia en ciertos taxones de animales sugerida por estudios previos, indican que quedan muchas preguntas sin respuesta sobre su biología. Al igual que otras bacterias intracelulares obligadas, son difíciles de cultivar fuera de su hospedero y son a menudo analizadas mediante métodos moleculares. Los ensayos de reacción en cadena de la polimerasa (PCR) se han desarrollado previamente para la detección de Wolbachia dentro de especies hospederas, y se han explorado varios genes para la tipificación de cepas y la reconstrucción filogenética. Sin embargo, dada la amplia gama de hospederos y la complejidad biológica de las relaciones simbióticas entre Wolbachia y su especie hospedera, el tener nuevos métodos podrían ayudar a acelerar el paso de la investigación de Wolbachia. Como parte de un objetivo general para estudiar la distribución de Wolbachia en mosquitos locales y en el gusano del corazón, Dirofilaria immitis (Leidy) (Rhabditida: Onchocercidae), nuestro objetivo fue el desarrollar métodos rentables que se puedan utilizar en la identificación y análisis de cepas. Desarrollamos un ensayo de PCR novedoso dirigido al gen gyrA de Wolbachia y exploramos varios métodos de preparación de muestras. Se detectaron presuntas Wolbachia en muestras de mosquitos de varios géneros, así como en muestras de D. immitis obtenidas de necropsias caninas. El análisis de la secuencia de ADN de los productos de la PCR confirmó la identidad de Wolbachia y reveló variabilidad dentro de algunas regiones del gen gyrA que corresponden a la especie hospedera. En consecuencia, este gen podría ser útil para futuros estudios filogenéticos y poblacionales.

Key Words: heartworm; symbiosis; Mansonia; evolution; mosquito; parasite

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Published

2021-01-17

Issue

Vol. 103, No. 4 (December 2020)

Section

Research Papers

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