Phylogenomic analysis of the Ponticus clade: strains isolated from the spotted rose snapper (Lutjanus guttatus).

The family Vibrionaceae is classified into many clades based on their phylogenetic relationships. The Ponticus clade is one of its clades and consists of four species, Vibrio panuliri, V. ponticus, V. rhodolitus, and V. taketomensis. Two strains, CAIM 703 and CAIM 1902, were isolated from the diseased spotted rose snapper external lesion (Lutjanus guttatus), they were analyzed to determine their taxonomic position, a phylogenetic analysis was performed based on the 16S rRNA sequences proved that the two strains are members of the genus Vibrio and they belong to the Ponticus clade. Then, a phylogenomic analysis was performed with four type strains and four reference strains isolated from marine organisms and aquatic environments. Multilocus Sequence Analysis (MLSA) of 139 single-copy genes showed that CAIM 703 and CAIM 1902 belong to V. panuliri. The 16S rRNA sequence similarity value between CAIM 703 and CAIM 1902 was 99.61%. The Ponticus clade species showed Average Nucleotide Identity (ANI) values between 78 to 80% against the two strains for ANIb, except V. panuliri LBS2T (99% and 100% similarity). Finally, this analysis represents the first phylogenomic analysis of the Ponticus clade where V. panuliri strains are reported from Mexico.

First report of Pectobacterium polaris causing aerial stem rot of potato in Mexico.

In Mexico, potato (Solanum tuberosum L.) is one of the most important vegetable crops for local consumption and industry. More than 1.8 million tons of potatoes are produced annually, of which the state of Sinaloa contributes with 21.5% (SIAP. 2022). In January 2020, potato plants (cv. FL1867) showing aerial stem rot symptoms were observed in a commercial field from the Santa Rosa Valley, in Northern Sinaloa with an incidence of 36%. Putative pectolytic bacteria showing pitting on crystal violet pectate (CVP) plates were restreaked and purified onto Nutritive Agar (NA) medium at 28°C. Four independent isolates were obtained (L25F-83, L25F-105, L25F-115, and L25F-125) from four symptomatic stems with biochemical and morphological characteristics related to Pectobacterium, such as catalase positive, oxidase negative, pectinolytic activity, Gram-negative and non-fluorescent in B-King medium. Bacterial gDNA was used for amplification and sequencing of two housekeeping genes (dnaX and leuS) (Portier et al. 2019). The nucleotide sequence identity between our isolates was 100% with both housekeeping genes (dnaX, OP376536-OP376539 and leuS, OP376540-OP376543). The BLASTn analysis of dnaX gene shared 98.98% and 99.19% identity with two soft-rot-causing bacterial strains NIBIO1006T (CP017481) and NIBIO1392 (CP017482) of Pectobacterium polaris, respectively; and with leuS gene shared 99.56% identity with P. polaris type strain NIBIO1006T. To further validate the identification, two strains, S5 (isolate L25F-105) and S6 (L25F-125), were selected for whole genome sequencing (WGS). The ANI values for closely related species were calculated using the Orthologous Average Nucleotide Identity (Ortho-ANI) Software Tool (OAT) (Lee et al. 2016). The Type (Strain) Genome Server (TYGS) was used for accurate genome-based taxonomy (https://tygs.dsmz.de) (Meier-Kolthoff et al. 2019). The genomes of P. polaris strains S5 (4811345 pb, GC=52%, AULSZ000000000) and S6 (4809754 pb, GC=52%, JAULTA000000000) revealed 96.86% and 96.07% Ortho-ANI and 73.6% and 66.5% dDDH with P. polaris type strain NIBIO1006T and P. parvum strain CFBP8630, respectively. The MLSA was performed on concatenated complete sequences of dnaX (OR470476, OR470477), leuS (OR470484, OR470485), recA (OR470488, OR470488), acnA (OR470474, OR470475), gapA (OR470478, OR470479), gyrA (OR470480, OR470481), icdA (OR470482, OR470483), proA (OR470486, OR470487), and rpoA genes (OR470490, OR470491). The consensus tree, constructed using the maximum likelihood method (MEGA 7.0), clustered strains S5 and S6 with P. polaris strains NIBIO1006T and NIBIO1392. The four isolates resulted pathogenic in tuber slices and potato seedlings (cv. Fianna) 24 and 72 h, respectively, after being inoculated with 30 µL bacterial suspension (1 X 108 CFU/ml) and incubated at 28 °C and 85% relative humidity. Bacterial colonies were reisolated from the affected tissue and identified with the same PCR primers as described above. Accordingly, P. polaris isolates S5 and S6, fulfill Koch's postulates for aerial stem rot of potato. To our knowledge, this is the first report of P. polaris causing aerial stem rot of potato in Mexico. This bacterium could be a significant threat to the local potato producers; therefore, an accurate and sensitive method of detection and epidemiological studies are needed to support an effective disease diagnosis and management program.

Vibrio eleionomae sp. nov., isolated from shrimp (Penaeus vannamei) pond water.

A novel Vibrio strain (CAIM 722T=SW9T=DSM 24596T) was isolated in 2003 from water of a shrimp (Penaeus vannamei) culture pond located in Los Mochis, Sinaloa, Mexico, and taxonomically characterized using a polyphasic approach. The 16S rRNA gene sequence clustered within those of the genus Vibrio, showing high similarity to the type strains of the Porteresiae clade. Multilocus sequence analysis using eight housekeeping genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, topA and 16S rRNA) and phylogenetic analysis with 139 single-copy genes showed that the strain forms an independent branch. Whole genome sequencing and genomic analyses (average nucleotide identity, OrthoANI, average amino acid identity and in silico DNA-DNA hybridization) produced values well below the thresholds for species delineation with all methods tested. In addition, a phenotypic characterization was performed to support the description and differentiation of the novel strain from related taxa. The results obtained demonstrate that the strain represent a novel species, for which the name Vibrio eleionomae sp. nov. is proposed.

Veronia nyctiphanis gen. nov., sp. nov., Isolated from the Stomach of the Euphausiid Nyctiphanes simplex (Hansen, 1911) in the Gulf of California, and Reclassification of Enterovibrio pacificus as Veronia pacifica comb. nov.

The bacterial strain 42Xb2 T was isolated from a female adult krill Nyctiphanes simplex infected with the apostome parasitoid ciliate Pseudocollinia brintoni in January 2007 in the Gulf of California. The strain has the morphological, phenotypic, and molecular characteristics of the bacteria of the family Vibrionaceae. The 16S rRNA gene sequence has a similarity of 97.7% with Enterovibrio pacificus SW014 T and 96.1% similarity with Enterovibrio norvegicus LMG 19839 T. A phylogenomic and a multilocus sequence analyses placed this strain close to the genera Enterovibrio, Grimontia, and Salinivibrio, but clearly forming a separate branch from these bacterial genera. Genomic analyses presented further support this result. A novel genus Veronia gen. nov. and a species Veronia nyctiphanis sp. nov. is here described with CAIM 600 T (= DSM 24592 T = CECT 7578 T) as the type strain. Morphological, physiological, and genetic evidence presented here support the unification of Enterovibrio pacificus and Veronia nyctiphanis in the new genus Veronia. Enterovibrio pacificus is reclassified as Veronia pacifica. V. pacifica is assigned as the type species of the new genus Veronia.Genome Sequencing Data The GenBank/EMBL/DDBJ accession numbers for the genome sequence of Veronia nyctiphanis CAIM 600 T is PEIB01 and of Enterovibrio pacificus CAIM 1920 T is LYBM01. The 16S rRNA gene sequence of V. nyctiphanis CAIM 600 T is JX129353.

Reproductive aspects of Grapsus grapsus (Decapoda: Grapsidae) on islands of the southeastern Gulf of California / Aspectos reproductivos de Grapsus grapsus (Decapoda: Grapsidae) en islas del sureste del Golfo de California

ABSTRACT Objective. The sex ratio, proportion of ovigerous females, length at sexual maturity and fecundity of Grapsus grapsus crabs in the Lobos, Venados and Pajaros islands (southeastern Gulf of California) were analyzed. Materials and methods. Sampling was conducted monthly between March 2011 and February 2012, during the night at low tide. Thirty crabs were collected in a quadrant (25 m2) at each sampling site on each island. cw (mm) and w (g) were determined. The sex ratio and size at sexual maturity (cw50%) were estimated, and for ovigerous females, embryonic stages and fecundity (gravimetric method) were determined. Results. The sex ratio (M:F) was 1:1.3. The average size at sexual maturity (cw50%) was 34.9 mm. The majority of females were ovigerous (71.3%), and 48% of the embryos of ovigerous females were at the red-orange phase. Egg diameter ranged from 1.1 to 5 µm, with an average of 2.05 µm. The mean fecundity was 24339.3 eggs. The maximum and minimum weight of ovigerous females was 69.9 and 15.2 g. Conclusions. The studied characteristics of sex ratio, proportion of ovigerous females, length at sexual maturity and fecundity of G. grapsus, indicate the effective administration and management of this resource in this area.

RESUMEN Objetivo. Se analizó la proporción de sexos, hembras ovígeras, talla de primera madurez sexual y fecundidad del cangrejo roca Grapsus grapsus en islas Lobos, Venados y Pájaros (sureste del Golfo de California). Material y métodos. Los muestreos fueron mensuales entre marzo 2011 y febrero 2012, las colectas fueron nocturnas durante la bajamar, se obtuvieron en un cuadrante (25 m2) por isla 30 organismos al azar, se les determinó el AN (mm) y PT (g). Se estimó la proporción de sexos y talla de primera madurez sexual (AN50%), se analizaron en hembras grávidas, las fases embrionarias y la fecundidad (método gravimétrico). Resultados. La proporción de M:H fue 1:1.3. La talla media de primera madurez fue AN50% 34.9 mm. Es evidente la presencia de hembras ovígeras (71.3%) y todas las fases embrionarias, la fase rojo-naranja fue la mayor representada en 48%. La variación del diámetro del huevo fue 1.1 a 5 µm y el promedio de 3.05 µm. La fecundidad media fue 24339.3 cigotos. El máximo y mínimo peso de hembras ovígeras fue 69.9 y 15.2 g, respectivamente. Conclusiones. Con base a las características biológicas del recurso tales como la proporción de sexos, hembras ovígeras, talla de primera madurez sexual y fecundidad en la población de G. grapsus, representa un efecto favorable en su posterior administración y manejo de este recurso en esta zona.

The complete mitogenome of excavating sponge Thoosa mismalolli (Demospongiae, Tetractinellida, Thoosidae) from Northeastern Tropical Pacific.

The complete mitogenome of Thoosa mismalolli Carballo, Cruz-Barraza & Gómez, 2004 (Tetractinellida, Thoosidae) was sequenced. This is the first complete mitogenome of the suborden Thoosina and the third Tetractinellid so far. The mitochondrial genome of T. mismalolli was assembled based on reads obtained with the Illumina HiSeq platform. The length of complete mitogenome is 19,019 bp long and contained 14 protein-coding genes and 23 tRNA, with two tRNA genes. Phylogenetic reconstruction (maximum-likelihood) based on mitogenome of Tetractinellids, supports T. mismalolli as a sister group. This result is congruent with those obtained with molecular markers (CO1, 18S, and 28S), supporting the monophyletic status of Thoosa and providing additional molecular data in favor of the suborder Thoosina.

Genomic taxonomy of the Mediterranei clade of the genus Vibrio (Gammaproteobacteria).

The first genomic study of Mediterranei clade using five type strains (V. mediterranei, V. maritimus, V. variabilis, V. thalassae, and V. barjaei) and fourteen reference strains isolated from marine organisms, seawater, water and sediments of the sea was performed. These bacterial strains were characterised by means of a polyphasic approach comprising 16S rRNA gene, multilocus sequence analysis (MLSA) of 139 single-copy genes, the DNA G + C content, ANI, and in silico phenotypic characterisation. We found that the species of the Mediterranei clade formed two separate clusters based in 16S rRNA gene sequence similarity, MLSA, OrthoANI, and Codon and Amino Acid usage. The Mediterranei clade species showed values between 76 and 95% for ANIb, 84 and 95% for ANIm. The core genome consisted of 2057 gene families and the pan-genome of 13,094 gene families. Based on the genomic analyses performed, the Mediterranei clade can be divided in two clusters, one with the strains of V. maritimus, V. variabilis and two potential new species, and the other cluster with the strains of V. mediterranei, V. thalassae, and V. barjaei.

Photobacterium lucens sp. nov., Isolated from a Cultured Shrimp Penaeus vannamei.

Two bacterial strains were isolated from the hepatopancreas of a cultured shrimp (Penaeus vannamei) in Sinaloa, México. Their partial 16S rRNA gene sequences clustered within those of the genus Photobacterium, showing high similarity to the type strains of Photobacterium angustum and Photobacterium leiognathi, were 87.1% and 97.5%, respectively. Multilocus sequence analysis using eight housekeeping genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, topA and 16S rRNA) and phylogenetic analysis with 139 single-copy genes showed that the new strains form an independent branch whole genome sequencing and genomic analyses (average nucleotide identity, average amino acid identity, and in silico DNA-DNA hybridization) produced values well below the thresholds for species delineation with all methods tested. In addition, a phenotypic characterization was performed to support the description and differentiation of the novel strains from related taxa. The results obtained demonstrate that the two strains represent a novel species for which the name Photobacterium lucens sp. nov. is proposed.

Vibrio sonorensis sp. nov. isolated from a cultured oyster Crassostrea gigas.

Strain CAIM 1076T was isolated from a cultured oyster Crassostrea gigas in Puerto Peñasco, Sonora state, México. The strain was taxonomically characterised by means of a genomic approach, comprising 16S rRNA gene sequence analysis, multilocus sequence analysis (MLSA), the DNA G+C content and whole genome analyses (ANI and GGDC), and by phenotypic characterisation. Strain CAIM 1076T was found to be catalase and oxidase positive, and cells were observed to be motile and facultative anaerobic. Analysis of the almost-complete 16S rRNA gene sequence placed this strain within the genus Vibrio; closely related species were Vibrio maritimus, Vibrio variabilis, Vibrio proteolyticus, and Vibrio nigripulchritudo with similarity values of 98.9, 98.5, 98.1, and 98.0 %, respectively. MLSA of six housekeeping genes (ftsZ, gapA, gyrB, recA, rpoA and topA) was performed with the closely related species. A draft genome sequence of strain CAIM 1076T was obtained. The DNA G+C content of this strain was determined to be 44.5 mol %. The genomic similarity values with V. maritimus were 71.6 % (ANIb), 85.1 % (ANIm) and a GGDC value of 20.3 ± 2.3 %; with V. variabilis the genomic similarities were 71.8 % (ANIb), 85.4 % (ANIm) and 20.0 ± 2.3 % (GGDC); with V. proteolyticus, 71.6 % (ANIb), 84.1 % (ANIm) and 18.8 ± 2.2 % (GGDC); and with V. nigripulchritudo, 70.8 % (ANIb), 84.9 % (ANIm) and 20.5 ± 2.3 % (GGDC). These ANI and GGDC values are below the thresholds for the delimitation of prokaryotic species, i.e., 95-96 and 70 %, respectively. Phenotypic characters also showed differences with the closely related species analysed. The results presented here support the description of a novel species, for which the name Vibrio sonorensis sp. nov. is proposed, with strain CAIM 1076T (=CECT 9100T, =DSM 102190T) as the type strain.

Unique and conserved genome regions in Vibrio harveyi and related species in comparison with the shrimp pathogen Vibrio harveyi CAIM 1792.

Vibrio harveyi CAIM 1792 is a marine bacterial strain that causes mortality in farmed shrimp in north-west Mexico, and the identification of virulence genes in this strain is important for understanding its pathogenicity. The aim of this work was to compare the V. harveyi CAIM 1792 genome with related genome sequences to determine their phylogenic relationship and explore unique regions in silico that differentiate this strain from other V. harveyi strains. Twenty-one newly sequenced genomes were compared in silico against the CAIM 1792 genome at nucleotidic and predicted proteome levels. The proteome of CAIM 1792 had higher similarity to those of other V. harveyi strains (78%) than to those of the other closely related species Vibrio owensii (67%), Vibrio rotiferianus (63%) and Vibrio campbellii (59%). Pan-genome ORFans trees showed the best fit with the accepted phylogeny based on DNA-DNA hybridization and multi-locus sequence analysis of 11 concatenated housekeeping genes. SNP analysis clustered 34/38 genomes within their accepted species. The pangenomic and SNP trees showed that V. harveyi is the most conserved of the four species studied and V. campbellii may be divided into at least three subspecies, supported by intergenomic distance analysis. blastp atlases were created to identify unique regions among the genomes most related to V. harveyi CAIM 1792; these regions included genes encoding glycosyltransferases, specific type restriction modification systems and a transcriptional regulator, LysR, reported to be involved in virulence, metabolism, quorum sensing and motility.

Vibrio mexicanus sp. nov., isolated from a cultured oyster Crassostrea corteziensis.

A bacterial strain was taxonomically characterised by means of a genomic approach comprising 16S rRNA gene sequence analysis, multilocus sequence analysis (MLSA), the DNA G+C content, whole genome analyses (ANI and GGDC) and phenotypic characterisation. The strain CAIM 1540(T) was isolated from a cultured oyster Crassostrea corteziensis in La Cruz, Sinaloa state, México. The isolate was found to be catalase and oxidase positive, cells were observed to be motile, O/129-sensitive and facultatively anaerobic. The almost-complete 16S rRNA gene sequence placed this strain within the genus Vibrio; the closest related species were found to be Vibrio aestivus, Vibrio marisflavi, Vibrio maritimus and Vibrio variabilis with similarity values of 99.02, 97.05, 96.70, and 96.59 % respectively. MLSA of four housekeeping genes (ftsZ, gapA, recA, and topA) was performed with the closely related species. A draft genome sequence of strain CAIM 1540(T) was obtained. The DNA G+C content of this strain was determined to be 43.7 mol%.The ANI values with V. aestivus were 89.6 % (ANIb), 90.6 % (ANIm) and a GGDC value of 39.5 ± 2.5 % was obtained; with V. marisflavi the genomic similarities were 71.5 % (ANIb), 85.5 % (ANIm) and 20.2 ± 2.3 % (GGDC); with V. maritimus 72.6 % (ANIb), 85.7 % (ANIm) and 22.0 ± 2.0 % (GGDC); and with V. variabilis 72.6 % (ANIb), 85.8 % (ANIm) and 21.6 ± 1.6 % (GGDC). These ANI and GGDC values are below the threshold for the delimitation of prokaryotic species, i.e. 95-96 and 70 %, respectively. Phenotypic characters also showed differences with the closest related species analysed. The results presented here support the description of a novel species, for which the name Vibrio mexicanus sp. nov. is proposed, with strain CAIM 1540(T) (= CECT 8828(T), = DSM 100338(T)) as the type strain. In addition, we found that the recently described species Vibrio thalassae and Vibrio madracius might be a single species because the values of ANIb 95.8 %, ANIm 96.6 % and GGDC 70.2 ± 2.9 % are above the accepted species thresholds.

Vibrio crosai sp. nov., isolated from a cultured oyster Crassostrea gigas.

A motile, facultative anaerobic, marine bacterial isolate (CAIM 1437(T)) was obtained from a cultured oyster (Crassostrea gigas) in Sonora, México. The strain was studied by a phylogenetic analysis based on sequences of the 16S rRNA and five housekeeping genes, i.e. ftsZ, gapA, pyrH, recA, and topA. Comparison of the almost-complete 16S rRNA gene sequence with those of other type strains of the genus Vibrio showed a close relationship with the type strains of Vibrio orientalis and Vibrio rotiferianus, with similarity values ranging from 98.4 to 98.3 %, respectively. MLSA placed this strain within the Orientalis clade. The DNA-DNA hybridization value of strain CAIM 1437(T) with V. orientalis was 59 % and with V. rotiferianus 55 %. The DNA G+C content was determined to be 45.6 mol %. Phenotypic characteristics also showed differences with the species analysed. The results presented here support the description of a novel species, for which the name Vibrio crosai sp. nov. is proposed, with CAIM 1437(T) (= DSM 27145(T)) as the type strain.