Phylogenetic Analysis of Some Species of the Anopheles hyrcanus Group (Diptera: Culicidae) in China Based on Complete Mitochondrial Genomes.

Some species of the Hyrcanus group are vectors of malaria in China. However, the member species are difficult to identify accurately by morphology. The development of sequencing technologies offers the possibility of further studies based on the complete mitochondrial genome. In this study, samples of mosquitoes of the Hyrcanus group were collected in China between 1997 and 2015. The mitochondrial genomes of ten species of the Hyrcanus group were analyzed, including the structure and base composition, codon usage, secondary structure of tRNA, and base difference sites in protein coding regions. Phylogenetic analyses using maximum-likelihood and Bayesian inference were performed based on mitochondrial genes and complete mitochondrial genomes The mitochondrial genome of 10 Hyrcanus group members ranged from 15,403 bp to 15,475 bp, with an average 78.23% (A + T) content, comprising of 13 PCGs (protein coding genes), 22 tRNAs, and 2 rRNAs. Site differences between some closely related species in the PCGs were small. There were only 36 variable sites between Anopheles sinensis and Anopheles belenrae for a variation ratio of 0.32% in all PCGs. The pairwise interspecies distance based on 13 PCGs was low, with an average of 0.04. A phylogenetic tree constructed with the 13 PCGs was consistent with the known evolutionary relationships. Some phylogenetic trees constructed by single coding regions (such as COI or ND4) or combined coding regions (COI + ND2 + ND4 + ND5 or ND2 + ND4) were consistent with the phylogenetic tree constructed using the 13 PCGs. The phylogenetic trees constructed using some coding genes (COII, ND5, tRNAs, 12S rRNA, and 16S rRNA) differed from the phylogenetic tree constructed using PCGs. The difference in mitochondrial genome sequences between An. sinensis and An. belenrae was very small, corresponding to intraspecies difference, suggesting that the species was in the process of differentiation. The combination of all 13 PCG sequences was demonstrated to be optimal for phylogenetic analysis in closely related species.

High frequency of Voltage-gated sodium channel (VGSC) gene mutations in Aedes albopictus (Diptera: Culicidae) suggest rapid insecticide resistance evolution in Shanghai, China.

BACKGROUND: Dengue fever is an infectious disease that is imported into Shanghai, China and requires prevention and control measures. Controlling the vector Aedes albopictus through insecticide use is a key approach to dengue control. However, the rapid evolution of insecticide resistance in Ae. albopictus has raised concerns about the failure of dengue control efforts. Knockdown resistance (kdr) caused by point mutations in the voltage-gated sodium channel (VGSC) gene is a primary mechanism of pyrethroid resistance. In this study, we investigated the kdr mutations of Ae. albopictus in Shanghai and evaluated the trend in its evolution. METHODOLOGY/PRINCIPAL FINDINGS: We collected 17 populations of Ae. albopictus from 15 districts in Shanghai in 2020, extracted genomic DNA from individual mosquitoes, and amplified Domain II, III, and IV in VGSC using PCR. Following sequencing, we obtained 658 VGSC sequences. We detected the nonsynonymous mutations V1016G, I1532T, and F1534S/C/I, among which V1016G and F1534C/I were reported in Shanghai for the first time and F1534I was a novel mutant allele in Ae. albopictus. The overall mutation frequency was 84.65%, with individual mutation frequencies ranging from 46.81% to 100%, excluding the Fengxian District population, which had a frequency of 0%. The V1016G and I1532T mutation types accounted for 7.14% and 3.42%, respectively. The mutant allele at codon 1534 accounted for 63.98% of all mutations, including TCC/S (62.77%), TGC/C (1.06%), and ATC/I (0.15%). We identified and classified five intron types in Domain III by length, including A (83 bp, 12.07%), B (68 bp, 87.30%), C (80 bp, 0.16%), D (72 bp, 0.16%), and E (70 bp, 0.31%). Individuals with intron B had a significant mutation tendency at codon 1534 relative to intron A (chi-square test, p < 0.0001). We found no correlation between mutation frequency and the amount of pyrethroid used (Pearson correlation, p = 0.4755). CONCLUSIONS/SIGNIFICANCE: In recent years, kdr mutations in the Ae. albopictus population in Shanghai have rapidly evolved, as evidenced by an increase in mutation types and significantly increased mutation frequency. The F1534I/ATC mutant allele was found to be a novel mutation, F1534C/TGC was reported for the first time in Shanghai, and intron B in Domain III was significantly associated with mutation frequency at codon 1534. Continuous monitoring of resistance changes and strict regulation of insecticide use are required.

Genetic structure of Aedes albopictus (Diptera: Culicidae) populations in China and relationship with the knockdown resistance mutations.

BACKGROUND: Mosquito control is needed to prevent dengue fever, which is mainly spread by Aedes albopictus in China. Application of insecticides is one of the main mosquito control methods; however, this approach can fail due to the knockdown resistance (kdr) gene mutation that causes decreased sensitivity to insecticides in Ae. albopictus. The kdr mutation patterns among different regions in China differ significantly. However, the underlying mechanism and factors that influence kdr mutation remain unclear. To explore the potential influence of genetic background on the development of insecticide resistance in Ae. albopictus, we analyzed the genetic structure of Ae. albopictus populations in China and its correlation with major kdr mutations. METHODS: We collected Ae. albopictus from 17 sites in 11 provinces (municipalities) across China from 2016 to 2021 and extracted the genomic DNA from individual adult mosquitoes. We selected eight microsatellite loci for genotyping, and based on microsatellite scores, we estimated intraspecific genetic diversity, population structure, and effective population size. The association between the intrapopulation genetic variation and F1534 mutation rate was evaluated by the Pearson correlation coefficient. RESULTS: Based on variation analysis of the microsatellite loci of 453 mosquitoes representing 17 populations throughout China, more than 90% of the variation occurred within individuals, whereas only about 9% of the variation occurred among populations, indicating that field populations of Ae. albopictus are highly polymorphic. The northern populations tended to belong to gene pool I (BJFT 60.4%, SXXA 58.4%, SDJN 56.1%, SXYC 46.8%), the eastern populations tended to belong to pool III (SH 49.5%, JZHZ 48.1%), and the southern populations tended to belong to three different gene pools. Moreover, we observed that the greater the fixation index (FST), the lower the wild-type frequency of F1534 of VSGC. CONCLUSIONS: The degree of genetic differentiation among Ae. albopictus populations in China was low. These populations were divided into three gene pools, in which the northern and eastern pools are relatively homogeneous, while the southern gene pool is heterogeneous. The possible correlation between its genetic variations and kdr mutations is also noteworthy.

Mitochondrial COI and Cytb gene as valid molecular identification marker of sandfly species (Diptera: Psychodidae) in China.

The accurate identification of sandfly species is crucial because some species transmit medically significant diseases, including leishmaniasis, bartonellosis and sandfly fever. However, due to the high similarity of the external morphology in sandfly species, identification can only be performed using internal morphological characteristics after dissection, which is time consuming and requires highly experienced staff. Thus, the introduction of suitable molecular markers may solve these identification problems. This study screened suitable DNA barcodes to identify common sandfly species in China. The phlebotomine sandflies were collected from Sichuan, Henan and Hainan Provinces from 2014 to 2016. The species were identified by the morphological characteristics of the pharyngeal armature and spermatheca. The genomic DNA of sandfly was extracted individually, and mitochondrial DNA (mtDNA) cytochrome C oxidase subunit I (COI) and cytochrome B (Cytb) as well as the 18S subunit of ribosomal DNA (rDNA) were amplified using polymerase chain reaction (PCR). Additionally, intraspecific and interspecific differences (p-distance) were calculated to evaluate the feasibility of the three gene fragments as a DNA barcode. The phylogeny trees of all sandfly species in this study were constructed using neighbor joining (NJ) method. Six species were identified by the morphological features, belonging to Phlebotomus and Sergentomyia, as Ph. chinensis s. l., Ph. stantoni, Se. bailyi, Se. iyengari, Se. squamirostris, and Se. squamipleuris. Analysis based on three gene fragments revealed some degree of intraspecific polymorphism among these sandfly species in China. The largest intraspecific variation occurred in Ph. chinensis s. l. (mtDNA COI, p-distance = 0.042; mtDNA Cytb, p-distance = 0.071), but the 18S rDNA fragment showed a small variation (p-distance = 0.005). The ranges of interspecific p-distances for mtDNA COI and mtDNA Cytb were 0.138 - 0.231 and 0.128 - 0.274, respectively. However, the interspecific p-distances of 18S rDNA are relatively low ranging from 0.003 to 0.055. Both mitochondrial COI and Cytb gene fragments are valid molecular identification markers in theses sandfly species. The topological structure of phylogeny trees based on mtDNA COI, mtDNA Cytb and 18S rDNA genes were all consistent with morphological classification. And we also found there were significant intraspecies differences within Ph. chinensis s. l. (0.006-0.071) and Se. bailyi (0.002-0.032) based on mtDNA Cytb gene fragment. Sequence alignment data suggested that Ph. chinensis s. l. from Sichuan should be Ph. sichuanensis, and the sandfly specimen collected from Henan was Ph. chinensis s. s.. There could be cryptic species in Se. bailyi from China.

The pattern of kdr mutations correlated with the temperature in field populations of Aedes albopictus in China.

BACKGROUND: Aedes albopictus is the primary vector of dengue fever in China. This mosquito species has a wide distribution range in China and can be found in the tropical climate zones of southern provinces through to temperate climate zones of northern provinces. Insecticides are an important control method, especially during outbreaks of dengue fever, but increasing insecticide resistance raises the risk of failure to control vector-borne diseases. Knockdown resistance (kdr) caused by point mutations in the voltage-gated sodium channel (VGSC) gene is a key mechanism that confers resistance to pyrethroids. In this study we explored the characteristics and possible evolutionary trend of kdr mutation in Ae. albopictus based on analysis of the kdr mutations in field populations of mosquitoes in China. METHODS: A total of 1549 adult Ae. albopictus were collected from 18 sites in China from 2017 to 2019 and 50 individuals from three sites in the 1990s. A fragment of approximately 350 bp from part of the S6 segment in the VGSC gene domain III was amplified and sequenced. Using TCS software version 1.21A, we constructed haplotypes of the VGSC gene network and calculated outgroup probability of the haplotypes. Data of annual average temperatures (AAT) of the collection sites were acquired from the national database. The correlation between AAT of the collection site and the kdr mutation rate was analyzed by Pearson correlation using SPSS software version 21.0. RESULTS: The overall frequency of mutant allele F1534 was 45.6%. Nine mutant alleles were detected at codon 1534 in 15 field populations, namely TCC/TCG (S) (38.9%), TTG/CTG/CTC/TTA (L) (3.7%), TGC (C) (2.9%), CGC (R) (0.3%) and TGG (W) (0.1%). Only one mutant allele, ACC (T), was found at codon 1532, with a frequency of 6.4% in ten field populations. Moreover, multiple mutations at alleles I1532 and F1534 in a sample appeared in five populations. The 1534 mutation rate was significantly positively related to AAT (Pearson correlation: r(18) = 0.624, P = 0.0056), while the 1532 mutation rate was significantly negatively related to AAT (Pearson correlation: r(18) = - 0.645, P = 0.0038). Thirteen haplotypes were inferred, in which six mutant haplotypes were formed by one step, and one additional mutation formed the other six haplotypes. In the samples from the 1990s, no mutant allele was detected at codon 1532 of the VGSC gene. However, F1534S/TCC was found in HNHK94 with an unexpected frequency of 100%. CONCLUSIONS: Kdr mutations are widespread in the field populations of Ae. albopictus in China. Two novel mutant alleles, F1534W/TGG and F1534R/CGC, were detected in this study. The 1534 kdr mutation appeared in the population of Ae. albopictus no later than the 1990s. The F1534 mutation rate was positively correlated with AAT, while the I1532 mutation rate was negatively correlated with AAT. These results indicate that iInsecticide usage should be carefully managed to slow down the spread of highly resistant Ae. albopictus populations, especially in the areas with higher AAT.

Congenital Atresia of Uterine Isthmus: Successful Diagnosis and End-To-End Anastomosis.

BACKGROUND: Müllerian duct anomalies are rare and occasionally diagnosed in adolescents with primary amenorrhea, abdominal pain, and sexual difficulties. They are present in a variety of forms and sometimes difficult to appropriately classify. The management of malformations remains controversial. CASE: A 15-year-old girl with primary amenorrhea and cyclic lower abdominal pain was found on laparoscopic examination to have an asymmetric ball-shaped uterus with isthmus stenosis suspended in the pelvis. The junction between the lower segment of uterus and the cervix was very thin and stenotic with scar-like tissue changes. Combined with pathologic evaluation, it was finally diagnosed as congenital atresia of uterine isthmus. Thus, an end-to-end anastomosis was performed instead of surgical resection. SUMMARY AND CONCLUSION: Müllerian duct anomalies in a variety of forms can be difficult to diagnosis correctly and treat appropriately. Preservation of reproductive ability is the first objective of all treatments.

Nearly Normal Congenital Cervical Fragmentation: A Hard-To-Diagnose and Successful End-To-End Anastomosis.

BACKGROUND: Congenital cervical fragmentation is a very rare genital tract malformation that usually presents in adolescence with primary amenorrhea and cyclic, monthly, lower abdominal pain. We report a nearly normal case of congenital cervical fragmentation and successful end-to-end anastomosis. CASE: A 15-year-old girl presented with primary amenorrhea with cyclic, monthly lower abdominal pain lasting for 15 months without any abnormal imaging findings (pelvic CT scan, ultrasonography, and hysteroscopy). Misdiagnosis and appendectomy was performed at the time of the initial lower abdominal pain. Diagnostic combined hysteroscopy and laparoscopy were performed in our hospital, and cervical fragmentation was diagnosed. A converted laparotomy end-to-end anastomosis was performed successfully, and regular menstruation was restored after the operation. SUMMARY AND CONCLUSION: The malformation of nearly normal congenital cervical fragmentation is existent and hard to diagnose. As long as the patient has persistent primary amenorrhea with cyclic, monthly lower abdominal pain, even if no abnormal findings on imaging, obstructive malformation of the reproductive duct should be the primary suspicion. Diagnosis and treatment should occur as early as possible to preserve the patient's fertility. End-to-end anastomosis is the best method for this type of patient.

Elevated CA125 in primary peritoneal serous psammocarcinoma: a case report and review of the literature.

Psammocarcinoma is a rare form of serous carcinoma of the ovary or peritoneum, and it is characterised by extensive psammoma body formation and invasion of surrounding structures. This report describes the case of a 42-year-old woman who presented with large ascites and raised CA125 level. Following a full staging laparotomy, she was made stable in stage IIIC. Despite the limited number of cases reported, the clinical prognosis of carcinomas resembling the serous borderline lesions seems much more favourable than the common serous carcinomas. A summary of all the reported cases is provided to highlight the clinical and prognostic features of this scarce tumour.