Prevalence, risk factors and survival in dogs with myxomatous valve degeneration / [Prevalência, fatores de risco e sobrevivência em cães com degeneração mixomatosa valvar]

The objective of this study was to describe the prevalence, survival time, and risk factors of myxomatous mitral valve disease (MMVD) in dogs by a retrospective observational cohort study design of a Brazilian veterinary center, admitted from 2012 to 2018 (2.585 days). Considering the 105 dog files confirmed as MMVD, the prevalence of the disease was higher in small-breed dogs with ages from 10 to 19 years (30.3%). The survival time of the dogs at B1 stage (1.854 ± 145 days) was higher than the B2 (1.508+209) and C/D (930 ± 209). Higher risks of death (hazard ratio) were detected in the presence of syncope (2.5), pulmonary crackling (2.0), dyspnea (1.9), and higher values of vertebral heart scale (1.7), radiographic left atrium dimension (1.8), the ACVIM staging (1.7), and the duration of QRS complex (1.05). The results achieved demonstrated higher prevalence of MMVD in old small-breed dogs, associated with longer survival time in the B1 stage of the disease, and higher risk of death related to the presence of clinical signs, such as dyspnea, syncope, and pulmonary crackling, beyond some radiographic, echocardiographic, and electrocardiographic variables of heart or atrial enlargement.(AU)

O objetivo deste estudo foi descrever a prevalência, o tempo de sobrevivência e os fatores de risco da doença mixomatosa valvar mitral (DMVM) em cães, por meio de estudo de coorte observacional retrospectivo, os quais foram atendidos em um centro veterinário de 2012 a 2018 (2.585 dias). Considerando os 105 arquivos de cães com confirmação da DMVM, maior prevalência foi observada em raças pequenas com idades entre 10 e 19 anos (30,3%). O tempo de sobrevivência de cães em estágio B1 (1.854+145 dias) foi superior ao B2 (1.508+209) e ao C/D (930+209). Maior risco de morte (hazard ratio) foi observado na presença de síncope (2,5), crepitação pulmonar (2,0), dispneia (1,9), bem como de valores superiores de VHS (1,7), dimensão radiográfica do átrio esquerdo (1,8), estadiamento ACVIM (1,7) e duração do complexo QRS (1,05). Os resultados obtidos demonstraram maior prevalência de DMVM em cães idosos de raças pequenas, associada a maior tempo de sobrevida no estágio B1 da doença e a maior risco de morte relacionado à presença de sinais clínicos como dispneia, síncope e crepitação pulmonar, além de algumas variáveis radiográficas, ecocardiográficas e eletrocardiográficas de cardiomegalia ou aumento atrial esquerdo.(AU)

Caste-specific gene expression underlying the differential adult brain development in the honeybee Apis mellifera.

Apis mellifera adult workers feature more developed key brain regions than queens, which allows them to cope with the broad range of duties they need to perform in a colony. However, at the end of larval development, the brain of queens is largely more developed than that of workers. Major morphogenetic changes take place after metamorphosis that shift caste-specific brain development. Here, we tested the hypothesis that this phenomenon is hormonally governed and involves differential gene expression. Our molecular screening approach revealed a set of differentially expressed genes in Pp (first pharate-adult phase) brains between castes mainly coding for tissue remodelling and energy-converting proteins (e.g. hex 70a and ATPsynß). An in-depth qPCR analysis of the transcriptional behaviour during pupal and pharate-adult developmental stage in both castes and in response to artificially augmented hormone titres of 18 genes/variants revealed that: i. subtle differences in hormone titres between castes might be responsible for the differential expression of the EcR and insulin/insulin-like signalling (IIS) pathway genes; ii. the morphogenetic activity of the IIS in brain development must be mediated by ILP-2, iii. which together with the tum, mnb and caspase system, can constitute the molecular effectors of the caste-specific opposing brain developmental trajectories.

Association of spontaneous abortion and Ureaplasma parvum detected in placental tissue.

Spontaneous abortion is considered a public health problem having several causes, including infections. Among the infectious agents, bacteria of the vaginal microbiota and Ureaplasma parvum have been associated with abortion, but their participation needs to be further elucidated. This study aims to evaluate the influence of Mollicutes on the development of spontaneous abortion. Women who underwent spontaneous abortion and those with normal birth (control) were studied. Samples of cervical mucus (CM) and placental tissue were collected to identify Mollicutes using the quantitative polymerase chain reaction methodology. Eighty-nine women who had a miscarriage and 20 women with normal pregnancies were studied. The presence of Mollicutes in placental tissue increased the chance of developing miscarriage sevenfold. The prevalence of U. parvum in women who experienced spontaneous abortion was 66.3% in placental tissue. A positive association was observed between the detection of U. parvum in samples of placental tissue and abortion. There was a significant increase in microbial load in placental tissue for M. hominis, U. urealyticum and U. parvum compared to the control group. Detection of U. parvum in CM in pregnant women can ascend to the region of the placental tissue and trigger a spontaneous abortion.

Cytotaxonomy of Dipetalogaster maxima Uhler, 1894 (Hemiptera, Reduviidae, Triatominae) / Citotaxonomia de Dipetalogaster maxima Uhler, 1894 (Hemiptera, Reduviidae, Triatominae)

Abstract The Triatomini tribe consists of ten genera and is regarded as one of the most important tribes from epidemiological point of view. The genus Dipetalogaster Usinger, 1939 is composed only by the species Dipetalogaster maxima Uhler, 1894. This triatomine is exclusive of the Mexico and is a potential vector for Chagas disease. Besides the epidemiological importance, the insects of the Triatominae subfamily are important biological models for cytogenetic studies. Therefore, in order to contribute to the knowledge on the reproductive biology and assist in citotaxonomy of D. maxima, this study aimed to describe spermatogenesis, as well as confirm the karyotype and heterochromatic patterns of this Mexican triatomine species. The seminiferous tubules were torn, fixed to a cover slip and underwent the cytogenetic technique of Lacto-acetic orcein and C-banding. Through the cytogenetics analysis of testicular material D. maxima it was possible to confirm the karyotype (2n = 22), describe the stages of spermatogenesis and characterize the heterochromatic pattern (restricted to sex chromosome Y) of the species. D. maxima showed the same arrangement of heterochromatin described for Triatoma lecticularia (Stål, 1859) (a species that occur in United States of American and Mexico and is phylogenetically related with D. maxima), highlighting the importance of this analysis as an optimization tool to explore phylogenetic correlations.

Resumo A tribo Triatomini consiste em dez gêneros e é considerada uma das tribos mais importantes do ponto de vista epidemiológico. O gênero Dipetalogaster Usinger, 1939 é composto apenas pela espécie Dipetalogaster maxima Uhler, 1894. Este triatomíneo é exclusivo do México e é um vetor potencial da doença de Chagas. Além da importância epidemiológica, os insetos da subfamília Triatominae são importantes modelos biológicos para estudos citogenéticos. Portanto, a fim de contribuir para o conhecimento da biologia reprodutiva e complementar o conceito específico de D. maxima, este trabalho objetivou descrever a espermatogênese, bem como confirmar o padrão cariotípico e heterocromático desta espécie mexicana, com foco citotaxonômico. Os túbulos seminíferos foram dilacerados, fixados em uma lamínula e submetidos à técnica citogenética de Orceína lacto-acética e Bandamento-C. Por meio da análise citogenética do material testicular de D. maxima foi possível confirmar o cariótipo (2n = 22), descrever os estágios da espermatogênese e caracterizar o padrão heterocromático (restrito ao cromossomo Y sexual) da espécie. D. maxima apresentou o mesmo arranjo de heterocromatina descrito para Triatoma lecticularia (Stål, 1859) (espécie que ocorre no México e nos Estados Unidos da América, filogeneticamente relacionada com D. maxima), destacando a importância desta técnica como ferramenta para explorar correlações filogenéticas.

The utilization of saliva as an early diagnostic tool for oral cancer: microRNA as a biomarker.

Recently, dentistry presents a preventive philosophy, seeking early diagnoses and minimally traumatic treatments for patients. Cancer is known for its aggressive nature, where its signals and symptoms may only appear in advanced stages of the disease, therefore, reducing the possibility of using atraumatic treatment options and patient survival. Saliva has in its composition substances which can be used as biomarkers for disease diagnoses, one of those being microRNA. microRNAs are a group of small RNA molecules with 18-24 nucleotides which have functions such as the degradation of oncogenes transcripted mRNA. The aim of this paper is to explore all theoretical possibilities that microRNA offers as an early diagnostic tool for oral cancer. Studies show that microRNA can be directly linked with cancer gene regulation. Because microRNA is more specific to tissues and diseases than mRNA, it holds the premise of being a feasible, non-invasive, and stable biomarker for early diagnosis of oral cancer. The fact that miRNA can be found in saliva makes it an extremely affordable and feasible option as a biomarker to be used. Since it is linked to regulating functions of cancer genes, it also brings hope that in the near future, it could be used as a reliable biomarker.

Cytotaxonomy of Dipetalogaster maxima Uhler, 1894 (Hemiptera, Reduviidae, Triatominae).

The Triatomini tribe consists of ten genera and is regarded as one of the most important tribes from epidemiological point of view. The genus Dipetalogaster Usinger, 1939 is composed only by the species Dipetalogaster maxima Uhler, 1894. This triatomine is exclusive of the Mexico and is a potential vector for Chagas disease. Besides the epidemiological importance, the insects of the Triatominae subfamily are important biological models for cytogenetic studies. Therefore, in order to contribute to the knowledge on the reproductive biology and assist in citotaxonomy of D. maxima, this study aimed to describe spermatogenesis, as well as confirm the karyotype and heterochromatic patterns of this Mexican triatomine species. The seminiferous tubules were torn, fixed to a cover slip and underwent the cytogenetic technique of Lacto-acetic orcein and C-banding. Through the cytogenetics analysis of testicular material D. maxima it was possible to confirm the karyotype (2n = 22), describe the stages of spermatogenesis and characterize the heterochromatic pattern (restricted to sex chromosome Y) of the species. D. maxima showed the same arrangement of heterochromatin described for Triatoma lecticularia (Stål, 1859) (a species that occur in United States of American and Mexico and is phylogenetically related with D. maxima), highlighting the importance of this analysis as an optimization tool to explore phylogenetic correlations.

Refluxo gastroesofágico em cadelas submetidas à ovário-histerectomia convencional ou videoassistida / Gastroesophageal reflux in dogs undergoing convencional or video-assisted ovariohysterectomy

A doença do refluxo gastroesofágico decorre do fluxo de conteúdo gastroduodenal para o esôfago e/ou órgãos adjacentes, o que leva à ampla gama de sinais e implicações clínicas. A incidência de refluxo gastroesofágico transoperatório em caninos é desconhecida. O objetivo deste trabalho foi, por meio da endoscopia flexível, avaliar a presença e quantificar o refluxo gastroesofágico em cadelas submetidas à ovário-histerectomia por duas abordagens cirúrgicas (convencional e videoassistida com dois portais), pré-medicadas com morfina. Cem por cento das cadelas submetidas à ovário-histerectomia videoassistida e 30% das cadelas submetidas à ovário-histerectomia convencional apresentaram algum grau de refluxo. A intensidade dos refluxos foi maior nas cadelas submetidas ao procedimento minimamente invasivo, visto que elas (10 entre 10 animais) apresentaram, no mínimo, um refluxo classificado em R4, enquanto as outras (três entre 10) apresentaram, no máximo, refluxos em grau R3, de acordo com a escala planejada para esta pesquisa. Concluiu-se que os procedimentos laparoscópicos sob o protocolo anestésico utilizado promovem mais refluxo gastroesofágico que os convencionais.(AU)

Gastroesophageal reflux disease occurs when gastric or duodenal contents flow back into the esophagus by retroperistalsis, which leads to several signs and clinical implications. The incidence of intraoperative gastroesophageal reflux in canines is unknown. The aim of this study was, using flexible endoscopy, to assess the presence and quantify gastroesophageal reflux in bitches undergoing ovariohysterectomy by two surgical approaches (conventional and video-assisted by two portals-access), pre-medicated with morphine. 100% of dogs submitted to video-assisted and 30% of dogs submitted to conventional ovariohysterectomy presented some reflux degree. Reflux intensity was higher in dogs submitted to the minimally invasive procedure, since they (10 out of 10 animals) had at least one reflux classified in R4 while the others (three out of 10) had a maximum reflux in degree R3 according to the planned scale for this research. The laparoscopic procedures under the used anesthetic protocol promote more gastroesophageal reflux than the conventional ones.(AU)

Refluxo gastroesofágico em cadelas durante ovário-histerectomia convencional submetidas a diferentes medicações pré-anestésicas / Gastroesophageal reflux in dogs undergoing conventional ovariohysterectomy using different premedications

A doença do refluxo gastroesofágico decorre do fluxo de conteúdo gastroduodenal para o esôfago e/ou para os órgãos adjacentes, o que leva à ampla gama de sinais e implicações clínicas. É desconhecida a incidência de refluxo gastroesofágico transoperatório em caninos. O objetivo deste trabalho foi, por meio da endoscopia flexível, avaliar a presença do refluxo gastroesofágico em cadelas submetidas a ovário-histerectomia com base nos fármacos analgésicos utilizados na medicação pré-anestésica (morfina, tramadol ou metadona). Concluiu-se que não houve diferença na incidência de refluxo gastroesofágico transoperatório, não tendo os fármacos testados influenciado de forma diferente esse comportamento; porém, alguns animais do grupo morfina apresentaram êmese pré-operatória. A gravidade dos refluxos foi maior nas cadelas submetidas ao uso da metadona, de acordo com o método de avaliação utilizado para esta pesquisa.(AU)

Gastroesophageal reflux disease arises from the gastroduodenal content flow to the esophagus and/or associated organs, which leads to the wide range of signs and clinical implications. Incidence of intraoperative gastroesophageal reflux disease in dogs is unknown. The objective was, through flexible endoscopy, to assess the presence of gastroesophageal reflux in dogs undergoing ovariohysterectomy based on analgesic drugs used in premedication (morphine, tramadol or methadone). It was concluded that there was no difference in the incidence of intraoperative gastroesophageal reflux, these drugs don't influence this behavior; however, in the morphine group some animals showed preoperative vomiting. The severity of the reflux was higher, but not significantly, in bitches undergoing the methadone according to the evaluator method used for this research.(AU)

Cytotaxonomy of Dipetalogaster maxima Uhler, 1894 (Hemiptera, Reduviidae, Triatominae)

Abstract The Triatomini tribe consists of ten genera and is regarded as one of the most important tribes from epidemiological point of view. The genus Dipetalogaster Usinger, 1939 is composed only by the species Dipetalogaster maxima Uhler, 1894. This triatomine is exclusive of the Mexico and is a potential vector for Chagas disease. Besides the epidemiological importance, the insects of the Triatominae subfamily are important biological models for cytogenetic studies. Therefore, in order to contribute to the knowledge on the reproductive biology and assist in citotaxonomy of D. maxima, this study aimed to describe spermatogenesis, as well as confirm the karyotype and heterochromatic patterns of this Mexican triatomine species. The seminiferous tubules were torn, fixed to a cover slip and underwent the cytogenetic technique of Lacto-acetic orcein and C-banding. Through the cytogenetics analysis of testicular material D. maxima it was possible to confirm the karyotype (2n = 22), describe the stages of spermatogenesis and characterize the heterochromatic pattern (restricted to sex chromosome Y) of the species. D. maxima showed the same arrangement of heterochromatin described for Triatoma lecticularia (Stål, 1859) (a species that occur in United States of American and Mexico and is phylogenetically related with D. maxima), highlighting the importance of this analysis as an optimization tool to explore phylogenetic correlations.

Resumo A tribo Triatomini consiste em dez gêneros e é considerada uma das tribos mais importantes do ponto de vista epidemiológico. O gênero Dipetalogaster Usinger, 1939 é composto apenas pela espécie Dipetalogaster maxima Uhler, 1894. Este triatomíneo é exclusivo do México e é um vetor potencial da doença de Chagas. Além da importância epidemiológica, os insetos da subfamília Triatominae são importantes modelos biológicos para estudos citogenéticos. Portanto, a fim de contribuir para o conhecimento da biologia reprodutiva e complementar o conceito específico de D. maxima, este trabalho objetivou descrever a espermatogênese, bem como confirmar o padrão cariotípico e heterocromático desta espécie mexicana, com foco citotaxonômico. Os túbulos seminíferos foram dilacerados, fixados em uma lamínula e submetidos à técnica citogenética de Orceína lacto-acética e Bandamento-C. Por meio da análise citogenética do material testicular de D. maxima foi possível confirmar o cariótipo (2n = 22), descrever os estágios da espermatogênese e caracterizar o padrão heterocromático (restrito ao cromossomo Y sexual) da espécie. D. maxima apresentou o mesmo arranjo de heterocromatina descrito para Triatoma lecticularia (Stål, 1859) (espécie que ocorre no México e nos Estados Unidos da América, filogeneticamente relacionada com D. maxima), destacando a importância desta técnica como ferramenta para explorar correlações filogenéticas.

Spermatogenesis in Nesotriatoma bruneri (Usinger 1944) (Hemiptera, Triatominae).

The Nesotriatoma genus consists of the species N. flavida N. bruneri and N. obscura, forming the Flavida complex. Variation in the size and morphological differences intraspecific of N. flavida led to the description of N. bruneri. Two years later, the same author proposed the synonymization of N. bruneri with N. flavida. Only in 1981 the specific status N. bruneri was recovered by means of morphological analysis of the genitalia. However, recently by genetic analysis, it was suggested that N. bruneri and N. flavida should be again synonymized. As Chagas disease has no cure, the main way to minimize the incidence of this disease is by vector control. Thus, grouping biological data from these hematophagous insects can assist in the development of vector control programs and mainly assist in taxonomic issues of synonymization. Thus, this paper describes spermatogenesis of N. bruneri. Three adult N. bruneri males were cytogenetically analyzed. The meiotic behavior observed for N. bruneri was very similar to that observed for the triatomine species with 23 chromosomes: during prophase, chromatin compaction was observed, the chromocenter composition was characterized (X1, X2 and Y), and the species karyotype was confirmed as 2n = 23 (20A + X1X2Y), as it was observed for N. flavida. Moreover, it was possible to observe anaphase and telophase. Thus, this study describes reproductive aspects of N. bruneri in order to contribute to the biological knowledge of these insects of epidemiological importance. Furthermore, this corroborates the synonymization of N. bruneri with N. flavida.

New chromosomal evidence for the origin of Triatoma infestans populations from Brazil.

In this study, the karyometry of different Triatoma infestans populations from different states of Brazil was analyzed and compared with those of a population from Cochabamba. No significant differences were found between the population from Cochabamba and those from Brazil. These results are consistent with the origin of the T. infestans populations of Brazil by a founder effect from Cochabamba. Moreover, these findings also confirm that a founder effect occurred during the dispersal of T. infestans populations in different Brazilian states.

Alkaline phosphatase activity in salivary gland cells of Rhodnius neglectus and R. prolixus (Hemiptera, Triatominae).

Alkaline phosphatase activity was detected in salivary gland cells of the Rhodnius neglectus Lent, 1954, and R. prolixus Stal, 1859, vectors of Trypanosoma cruzi Chagas, 1909 (etiological agent of Chagas disease) and T. rangeli Tejera, 1920 (pathogenic to insect). The Gomori technique was used to demonstrate alkaline phosphatase activity. Alkaline phosphatase activity was observed throughout the entire gland, with an increased activity in the posterior region of the principal gland. In particular, phosphatase activity was found in the nucleolar corpuscles, suggesting a relationship with the rRNA transcription and ribosomal biogenesis. Alkaline phosphatase was also detected in the nuclear membrane and nuclear matrix, suggesting an association with the nucleo-cytoplasmic transport of ribonucleoproteins and the mechanisms of cell cycle and DNA replication, respectively. This study highlights the importance of alkaline phosphatase in the salivary gland of R. prolixus and R. neglectus and emphasizes its importance in secretory activity. Secretory activity is directly involved in hematophagy and, consequently, in development during metamorphosis. The observed presence of alkaline phosphatase suggests its involvement in the production of saliva allowing feeding of these insects that are important vectors of Chagas disease.

Differentiation between Triatoma arthurneivai and Triatoma wygodzinskyi (Hemiptera: Reduviidae: Triatominae) using cytotaxonomy.

Using classic morphometric techniques to examine the head and thorax of Triatoma specimens, researchers identified a possible taxonomic problem involving T. arthurneivai (Lent & Martins) and T. wygodzinskyi (Lent). A recent geometric morphometric study indicated that the insects captured outside the Serra do Cipó region, State of Minas Gerais, Brazil, were T. wygodzinskyi. The misidentification of T. arthurneivai as T. wygodzinskyi could result in several problems associated with entoepidemiological lifting, the biological characterization of the species, and phylogenetic reconstruction. For the first time, we describe the use of cytogenetic analysis as a tool for differentiation between T. arthurneivai and T. wygodzinskyi. The results indicated that both species had the same number of chromosomes 2n = 22 (20A + XY). However, analyses of spermatocytes during early prophase indicated that it was possible to differentiate T. arthurneivai and T. wygodzinskyi, because only T. arthurneivai exhibited heteropycnotic blocks distributed in the chromatin. Therefore, we highlight the analysis of spermatocytes as a taxonomic tool for the characterization of T. arthurneivai and T. wygodzinskyi, and suggest that the technique can be used for entoepidemiological lifting in vector control programs. Thus, the results presented here, in conjunction with morphometric analyses, are of utmost taxonomic and epidemiological importance for the identification of T. arthurneivai and T. wygodzinskyi specimens.

New record and cytogenetic analysis of Psammolestes tertius Lent & Jurberg, 1965 (Hemiptera, Reduviidae, Triatominae) from Bahia State, Brazil.

This paper reports on the first occurrence of Psammolestes tertius in the Chapada Diamantina region, located in the city of Seabra, Bahia State, in northeastern Brazil. Following an active search, 24 P. tertius specimens were collected from Phacellodomus rufifrons (rufous-fronted thornbird) nests. The insects did not present any symptoms of infection by Trypanosoma cruzi. P. tertius males were cytogenetically analyzed, and the results were compared with those of other specimens from the Brazilian State of Ceará. Triatomines from both locations presented the same cytogenetic characteristics: 22 chromosomes, little variation in the size of the autosomes, Y chromosomes that were larger than the X chromosomes, a chromocenter formed only by the sex chromosomes during prophase, and autosomes lacking constitutive heterochromatin. However, it is important to note that this species shows intraspecific chromosomal variation. In light of the results obtained, it is recommended that more studies be performed to characterize P. tertius. These studies will be particularly helpful in understanding this species in ecological, biological, biogeographical, and phylogenetic terms.

Animal Mitochondrial DNA Replication.

Recent advances in the field of mitochondrial DNA (mtDNA) replication highlight the diversity of both the mechanisms utilized and the structural and functional organization of the proteins at mtDNA replication fork, despite the relative simplicity of the animal mtDNA genome. DNA polymerase γ, mtDNA helicase and mitochondrial single-stranded DNA-binding protein-the key replisome proteins, have evolved distinct structural features and biochemical properties. These appear to be correlated with mtDNA genomic features in different metazoan taxa and with their modes of DNA replication, although substantial integrative research is warranted to establish firmly these links. To date, several modes of mtDNA replication have been described for animals: rolling circle, theta, strand-displacement, and RITOLS/bootlace. Resolution of a continuing controversy relevant to mtDNA replication in mammals/vertebrates will have a direct impact on the mechanistic interpretation of mtDNA-related human diseases. Here we review these subjects, integrating earlier and recent data to provide a perspective on the major challenges for future research.

Description of the diploid chromosome set of Triatoma pintodiasi (Hemiptera, Triatominae).

Triatoma pintodiasi has been described and recently grouped in the Rubrovaria subcomplex. T. pintodiasi was initially compared to T. carcavalloi by staining and subsequently identified as T. circummaculata. However, after thorough examination, it was observed to be a cryptic species of T. circummaculata, and was described based on morphological features, morphometric data, and biochemical patterns of hemolymph. Thus, this paper aims to describe the karyotype of, and spermatogenesis in, T. pintodiasi, in order to elucidate the reproductive biology and taxonomy of the species. Sex chromosomes of T. pintodiasi formed a heteropyknotic chromocenter, and compaction of chromatin was observed during prophase. However, in contrast to observations in T. carcavalloi and T. circummaculata, in T. pintodiasi it was observed individualization of the sex chromosomes. The diploid chromosome set of the species 2n = 22 (20A + XY) is described through analysis of metaphases I and II. Initial cytogenetic characteristics of T. pintodiasi are described and the observed differences in the chromocenter are suggested as a possible cytotaxonomic tool. To gain a better understanding of the specific status of this cryptic species, however, we emphasize the need for further cytogenetic, molecular, biological, and biogeographical analysis, in addition to experimental hybrid crosses with other species of the Rubrovaria subcomplex.