Brevipalpus sp.

Ávalos-Cerdas, Juan Manuel, Otero-Colina, Gabriel, Ochoa-Martínez, Daniel L., Villegas-Monter, Ángel, Bautista-Martínez;, Néstor, Suárez-Espinosa, Javier, Carrillo-Benítez, María Guadalupe, Tassi, Aline Daniele, Ochoa, Ronald & Valdez Carrazco, Jorge Manuel, 2023, Characterization of an aberrant population of Brevipalpus californicus and its role as a vector of citrus leprosis viruses, Acarologia 63, pp. 45-68 : 47-49

Establishment of Brevipalpus sp. (Tetipac) colonies

Specimens of “ Brevipalpus sp. (Tetipac)” were collected in Muyuapan, municipality of

Tetipac, Guerrero, Mexico (18°37′32.81″N ; 99°41′56.79″W), an area considered free of

leprosis ( SENASICA, 2016). The host plants were sweet orange ( Citrus x sinensis (L.) Osbeck. ‘Valencia’) and Mexican lime [ Citrus x aurantifolia (Christm.) Swingle]. Once the mites were obtained, single-parent or isoline colonies were established from a single female, following the method described by García-Escamilla et al. (2017) on sour oranges. A single adult

female was placed on each orange and allowed to oviposit. After at least one egg was laid, the female was extracted to be mounted on a slide in Hoyer’s solution ( Krantz & Walter, 2009) for identification. Only one of the isolines could be established permanently, multiplied on several oranges and became the source of experimental material. About every two months, several specimens were taken from the infested oranges and mounted in temporary slides to confirm they were still morphologically distinguishable Brevipalpus as sp. (Tetipac).

The colony was incubated at 24–27°C and 40–50% relative humidity, with led illumination

of 14.76 µmol/m 2 s and 12:12 h light period. The replacement oranges were obtained from

the Campus Montecillo of the Colegio de Postgraduados and other nearby locations in the municipality of Texcoco, Mexico State (19°27′49.59″N, 98°54′19.92″W), which is also a

leprosis-free zone ( SENASICA, 2016). The oranges were previously cleaned as described by García-Escamilla et al. (2017).

Morphometric characterization

The morphological characterizations were performed for the two mite populations used for the virus transmission experiments: Brevipalpus sp. (Tetipac) and “ B. californicus (Texcoco) ”.

The specimens of B. californicus (Texcoco) were obtained from a colony established in 2016 from a female collected on sour orange in Texcoco de Mora, State of Mexico (19°27′46.4″N ; 98°54′14.75″W). Twenty adult females taken from each colony were mounted on slides with Hoyer’s solution ( Krantz & Walter, 2009). The slides were observed using both phase-contrast microscopy (Carl Zeiss GmbH Primo Star mod. 314800509) and differential interference contrast ( DIC) microscopy (photomicroscope 3, Carl Zeiss). The observed characteristics were compared against both the original description ( Baker, 1949) and redescriptions B of. lewisi ( Baker & Tuttle, 1987 ; Beard et al., 2012).

Thirty-seven measurements were taken for each specimen, using the software ImageJ

1.52a ( Table 1), the terminology of Beard et al. (2015) and Tassi (2018) was followed. Each measurement was taken by processing the photomicrographs with a Carl Zeiss photomicroscope

3, DIC optical microscope at magnifications from 640 to 1000X. For lengths, the scale obtained for each photograph was used. ImageJ 1.52 measures the roundness factor as an interval from

0 (completely unrounded) to 1 (perfect roundness); the arithmetical mean was estimated for each specimen. The slides used for this study are in the collection of the Acarology Laboratory of the Colegio de Postgraduados, Montecillo, State of Mexico, Mexico.

The same measurements were taken for 15 specimens from the type series of B. lewisi [hereafter, B. lewisi (type)] and five specimens of the type series of B. californicus , [hereafter B. californicus (type)] from photomicrographs of specimens located in the US National Mite collection held at the Systematic Entomology Laboratory, Beltsville Agricultural Research Station, USDA, United States of America. These photomicrographs were taken using DIC with a Carl Zeiss Axioplan 2 microscope and used for comparisons of the characteristics of Brevipalpus sp. (Tetipac) and B. californicus (Texcoco) . All the obtained images were edited using the GIMP 2.8.22 software.

The individual seta length was measured from the base to the apex, while the distance between setae was measured between their bases. The degree of the roundness character was measured using only the cells in the photographs with a defined edge in the ornamentations on the dorsal surface of the propodosoma as valid data. The well-defined cells were mostly confined to the sublateral area of the propodosomal plate. The number of measured cells per specimen were (mean ± standard error), Brevipalpus sp. (Tetipac), 84.57 ± 3.58; B. californicus (Texcoco) , 92.67 ± 7.84; B. lewisi (type), 55.3 ± 3.56; B. californicus (type), 89.75 ± 5.51.

The values of each variable were compared through analysis of variance ( ANOVA) and Tukey multiple comparison (α=0.05). The normality (Shapiro-Wilk test) and variance homogeneity (Bartlett test) assumptions were verified. For the joint analysis of all the variables, we used a multivariate analysis, where principal component analyses ( PCA) and a cluster analysis were performed. All the analyses were performed using the SAS v. 9.4 software.

Supplementary observations of Brevipalpus sp. (Tetipac) and B. californicus (Texcoco)

were done using SEM to illustrate the chaetotaxy of the idiosoma, the legs, the patterns of reticulation on the cuticle and the microplates. The samples were processed through dehydration in graduated ethanol series, critical point dried and coated in gold ( Tassi, 2018) for observation in Jeol JSM IT300 or Quanta FEG650 microscopes, or through freezing in liquid nitrogen and coating in platinum ( Bauchan et al., 2019) for observation in a Hitachi S-4700 microscope.

Molecular characterization