Elongases of Long-Chain Fatty Acids ELO2 and ELO9 Are Involved in Cuticle Formation and Function in Fecundity in the Yellow Fever Mosquito, Aedes aegypti.

Long-chain fatty acid elongases (ELOs) play important roles in the metabolism of fatty acids in insects. In this study, the genes for two elongases from Aedes aegypti were identified, AeELO2 and AeELO9. Quantitative real time PCR showed that AeELO2 and AeELO9 are expressed at all developmental stages and some body parts, but with different expression patterns. RNAi-mediated knockdown of AeELO2 and AeELO9 was performed to investigate their roles in the development, growth, osmotic balance, and cold tolerance of Ae. aegypti. Knockdown of AeELO2 slowed larval growth and development by causing molting abnormalities. Additionally, 33% ± 3.3% of adults died during oviposition, accompanied by an abnormal extension of cuticles in AeELO2-dsRNA knockdown mosquitos. Knockdown of AeEL09 resulted in abnormal balance of cuticular osmotic pressure and a reduction in egg production. The maximal mRNAs of AeELO2 and AeELO9 were detected in eggs at 72 h after oviposition. Moreover, AeELO2 knockdown reduced the egg hatching rates and AeELO9 knockdown larvae did not develop well. In summary, AeELO2 is involved in larval molting and growth, and its knockdown affects the flexibility and elasticity of adult mosquito cuticles. AeELO9 regulates cold tolerance, osmotic balance, and egg development in Ae. aegypti.

Identification of Theileria spp. and investigation of hematological profiles of their infections in goats in Hainan Island, China.

Theileria spp. are a group of parasites primarily transmitted by ticks and can pose a significant threat to domestic and wild animals globally. The main objective of this study was to understand the epidemiology of Theileria spp. in goats of Hainan Island/province, which is the only tropical region of China, and to study their hematological profiles in naturally infected goats. A total of 464 blood samples were collected from randomly selected local adult goats (Capra hircus, local domestic breed with black hair), from six cities and eight counties of Hainan, from November 2017 to October 2020. Blood smear microscopy of the sample and a nested polymerase chain reaction (nPCR) targeting the 18S rRNA gene combined with DNA sequencing were used to detect piroplasm infections in goats. Data analysis of the obtained sequences revealed that all the sequences were highly similar to the Theileria luwenshuni 18S rRNA gene sequence from the database. This result is consistent with the microscopic examination. In the hematological test, hematocrit, mean corpuscular volume, and mean corpuscular hemoglobin of the goats naturally infected with T. luwenshuni significantly increased, while mean corpuscular hemoglobin concentration and red blood cell distribution width (RDW) were significantly decreased. Results showed that T. luwenshuni could cause macrocytic, hypochromic anemia in goats. This study provides reliable and comprehensive information about the epidemiology of the parasite infections and hematological profile of the infected goats in Hainan, which encourages further investigations to develop practical control strategies for Theileria spp. infections in tropical areas.

TITLE: Identification de Theileria spp. et enquête sur les profils hématologiques de leurs infections chez les chèvres de l'île de Hainan, en Chine. ABSTRACT: Les Theileria spp. sont un groupe de parasites principalement transmis par les tiques qui peuvent constituer une menace importante pour les animaux domestiques et sauvages dans le monde. L'objectif principal de cette étude était de comprendre l'épidémiologie de Theileria spp. chez les chèvres de l'île/province de Hainan, qui est la seule région tropicale de Chine et étudier les profils hématologiques des chèvres naturellement infectées. 464 échantillons de sang ont été prélevés sur des chèvres adultes locales sélectionnées au hasard (Capra hircus, race domestique locale à poils noirs), dans 6 villes et 8 comtés de Hainan, de novembre 2017 à octobre 2020. L'étude microscopique du frottis sanguin de l'échantillon et la réaction en chaîne par polymérase nichée (nPCR) ciblant le gène de l'ARNr 18S combinée au séquençage de l'ADN ont été utilisées pour détecter les infections à piroplasmes chez les chèvres. L'analyse des séquences obtenues a révélé que toutes les séquences étaient très similaires à la séquence du gène de l'ARNr 18S de T. luwenshuni de la base de données. Le résultat est cohérent avec l'examen microscopique. Dans le test hématologique, l'hématocrite, le volume corpusculaire moyen et l'hémoglobine corpusculaire moyenne des chèvres naturellement infectées par T. luwenshuni ont augmenté de manière significative, tandis que la concentration moyenne d'hémoglobine corpusculaire et la largeur de distribution des globules rouges (RDW) ont été significativement diminuées. Les résultats ont montré que T. luwenshuni pouvait provoquer une anémie macrocytaire et une anémie hypochrome chez les chèvres. Cette étude fournit des informations fiables et complètes sur l'épidémiologie des infections parasitaires et le profil hématologique des chèvres infectées à Hainan, ce qui encourage des investigations supplémentaires pour développer des stratégies pratiques de contrôle des infections par Theileria spp. dans les zones tropicales.

[Research advances in assessment tools for feeding problems in children].

As one of the most important non-nutritional factors associated with children's growth and development, feeding problems in children are getting more and more attention from medical professionals and guardians. The evaluation of feeding problems has developed from the single-factor and descriptive research in the past to the multi-factor and analytical research at present, and thus a good quantitative analysis system is increasingly important for researchers. However, the development of localized quantitative analysis tools remains a weak link in this field. Therefore, it is a research hotspot to develop child feeding assessment scales and questionnaires with high reliability, validity, and operability in combination with China's cultural background and eating habits and provide effective assessment tools for feeding problems in Chinese children. Through classification based on research mode and screening, this article reviews the research findings in the field of child feeding, so as to provide a basis for future research.

Arylalkalamine N-acetyltransferase-1 functions on cuticle pigmentation in the yellow fever mosquito, Aedes aegypti.

Arylalkylamine N-acetyltransferase (aaNAT) catalyzes the acetylation of dopamine, 5-hydroxy-tryptamine, tryptamine, octopamine, norepinephrine and other arylalkylamines to form respective N-acetyl-arylalkylamines. Depending on the products formed, aaNATs are involved in a variety of physiological functions. In the yellow fever mosquito, Aedes aegypti, a number of aaNATs and aaNAT-like proteins have been reported. However, the primary function of each individual aaNAT is yet to be identified. In this study we investigated the function of Ae. aegypti aaNAT1 (Ae-aaNAT1) in cuticle pigmentation and development of morphology. Ae-aaNAT1 transcripts were detected at all stages of development with highest expressions after pupation and right before adult eclosion. Ae-aaNAT1 mutant mosquitoes generated using clustered regularly interspaced palindromic repeats (CRISPR) - CRISPR-associated protein 9 had no obvious effect on larval and pupal development. However, the mutant mosquitoes exhibited a roughened exoskeletal surface, darker cuticles, and color pattern changes suggesting that Ae-aaNAT1 plays a role in development of the morphology and pigmentation of Ae. aegypti adult cuticles. The mutant also showed less blood feeding efficiency and lower fecundity when compared with the wild-type. The mutation of Ae-aaNAT1 influenced expression of genes involved in cuticle formation. In summary, Ae-aaNAT1 mainly functions on cuticular pigmentation and also affects blood feeding efficiency and fecundity.

RNA interference of odorant receptor CquiOR114/117 affects blood-feeding behavior in Culex quinquefasciatus.

The odorant receptors (ORs) play a critical role for mosquitoes in the identification of blood-feeding hosts and other physiological processes. The OR8 subfamily in mosquitoes has been shown to be strongly involved in the detection the mammalian host associated odor, 1-octen-3-ol. CquiOR114/117 has been shown to be an orthologous OR8 in Culex quinquefasciatus Say. In this study, the expression of CquiOR114/117 in the different developmental stages of Cx. quinquefasciatus was detected by the amplification of CquiOR114/117 with real-time fluorescence quantitative polymerase chain reaction (PCR). RNA interference (RNAi) technology was used to interfere with the expression of CquiOR114/117 in females to observe the blood-feeding behavior change. The results showed that the expression level of CquiOR114/117 in the egg-to-pupa stage was significantly lower than that in the adult stage and that the expression level of the female mosquitoes peaked on the third day after emergence. The expression of CquiOR114/117 was significantly decreased in the 2-6 days after the injection of dsRNA compared with the control groups. The analysis of the blood-feeding behavior showed a significant positive correlation between CquiOR114/117 expression and the engorgement rate of the mosquitoes. CquiOR114/117 is speculated to have an effect on the blood-feeding behavior of Cx. quinquefasciatus.

RNA interference-mediated knockdown of 3, 4-dihydroxyphenylacetaldehyde synthase affects larval development and adult survival in the mosquito Aedes aegypti.

BACKGROUND: The cuticle is an indispensable structure that protects the mosquito against adverse environmental conditions and prevents pathogen entry. While most cuticles are hard and rigid, some parts of cuticle are soft and flexible to allow movement and blood-feeding. It has been reported that 3, 4-dihydroxyphenylacetaldehyde (DOPAL) synthase is associated with flexible cuticle formation in Aedes aegypti. However, the molecular function of DOPAL synthase in the ontogenesis of mosquito remains largely unknown. In this study, we characterized gene expression profiles of DOPAL synthase and investigated its functions in larvae and female adults of Aedes agypti by RNAi. RESULTS: Our results suggest that the expression of DOPAL synthase is different during development and the transcriptional level reached its peak at the female white pupal stage, and DOPAL synthase was more highly expressed in the cuticle and midgut than other tissues in the adult. The development process from larva to pupa was slowed down strikingly by feeding the first-instar larvae with chitosan/DOPAL synthase dsRNA nanoparticles. A qRT-PCR analysis confirmed that the dsRNA-mediated transcription of the DOPAL synthase was reduced > 50% in fourth-instar larvae. Meanwhile, larval molt was abnormal during development. Transmission electron microscopy results indicated that the formation of endocuticle and exocuticle was blocked. In addition, we detected that the dsDOPAL synthase RNA caused significant mortality when injected into the female adult mosquitoes. CONCLUSIONS: Our findings demonstrate that DOPAL synthase plays a critical role in mosquito larval development and adult survival and suggest that DOPAL synthase could be a good candidate gene in RNAi intervention strategies in mosquito control.

[Research proceedings on primate comparative genomics].

With the accomplishment of genome sequencing of human, chimpanzee and other primates, there has been a great amount of primate genome information accumulated. Primate comparative genomics has become a new research field at current genome era. In this article, we reviewed recent progress in phylogeny, genome structure and gene expression of human and nonhuman primates, and we elaborated the major biological differences among human, chimpanzee and other non-human primate species, which is informative in revealing the mechanism of human evolution.

Rapid evolution and copy number variation of primate RHOXF2, an X-linked homeobox gene involved in male reproduction and possibly brain function.

BACKGROUND: Homeobox genes are the key regulators during development, and they are in general highly conserved with only a few reported cases of rapid evolution. RHOXF2 is an X-linked homeobox gene in primates. It is highly expressed in the testicle and may play an important role in spermatogenesis. As male reproductive system is often the target of natural and/or sexual selection during evolution, in this study, we aim to dissect the pattern of molecular evolution of RHOXF2 in primates and its potential functional consequence. RESULTS: We studied sequences and copy number variation of RHOXF2 in humans and 16 nonhuman primate species as well as the expression patterns in human, chimpanzee, white-browed gibbon and rhesus macaque. The gene copy number analysis showed that there had been parallel gene duplications/losses in multiple primate lineages. Our evidence suggests that 11 nonhuman primate species have one RHOXF2 copy, and two copies are present in humans and four Old World monkey species, and at least 6 copies in chimpanzees. Further analysis indicated that the gene duplications in primates had likely been mediated by endogenous retrovirus (ERV) sequences flanking the gene regions. In striking contrast to non-human primates, humans appear to have homogenized their two RHOXF2 copies by the ERV-mediated non-allelic recombination mechanism. Coding sequence and phylogenetic analysis suggested multi-lineage strong positive selection on RHOXF2 during primate evolution, especially during the origins of humans and chimpanzees. All the 8 coding region polymorphic sites in human populations are non-synonymous, implying on-going selection. Gene expression analysis demonstrated that besides the preferential expression in the reproductive system, RHOXF2 is also expressed in the brain. The quantitative data suggests expression pattern divergence among primate species. CONCLUSIONS: RHOXF2 is a fast-evolving homeobox gene in primates. The rapid evolution and copy number changes of RHOXF2 had been driven by Darwinian positive selection acting on the male reproductive system and possibly also on the central nervous system, which sheds light on understanding the role of homeobox genes in adaptive evolution.

A novel fusion gene, TRIM5-Cyclophilin A in the pig-tailed macaque determines its susceptibility to HIV-1 infection.

OBJECTIVE: In Old World monkeys, the tripartite motif 5alpha (TRIM5alpha) protein confers resistance to HIV-1 infection following virus entry into host cells. However, the pig-tailed macaque (Macaca nemestrina) is an exception and is susceptible to HIV-1 infection. This study dissects the molecular mechanism of the pig-tailed macaque's susceptibility to HIV-1 infection. METHODS: Genomic sequencing and expression analysis of the TRIM5alpha gene was conducted in the pig-tailed macaque. A novel TRIM5-Cyclophilin A fusion gene isoform was identified and subsequently cloned into the pcDNA3.1(+) expression vector. This construct was transfected into HeLa-T4 or HeLa cells which were then infected with the HIV-1IIIB or HIV-GFP-VSVG pseudotyped virus, to examine the effects of the TRIM5-Cyclophilin A fusion protein on HIV-1 infection. RESULTS: A novel TRIM5-Cyclophilin A fusion gene (mnTRIMCyp) in the pig-tailed macaque was found and its fusion pattern is different from the known fusion gene in the owl monkey (owlTRIMCyp). TRIMCyp protein expression in transfected cells was confirmed by western blotting. The tests using HIV-1IIIB and HIV-GFP-VSVG pseudotyped virus indicated that mnTRIMCyp did not inhibit HIV-1 replication at various multiplicities of infection. CONCLUSIONS: The mnTRIMCyp fusion protein does not restrict replication of HIV-1, which provides a potential molecular mechanism that might explain why the pig-tailed macaque is prone to HIV-1 infection, the only known exception in Old World monkeys.

Adaptive evolution of primate TRIM5alpha, a gene restricting HIV-1 infection.

Recent studies showed that nonhuman primate TRIM5alpha can efficiently block HIV-1 infection in human cell lines. It can also restrict other retroviruses, therefore, suggested as a general defender against retrovirus infection. Here, we present an evolutionary analysis of TRIM5alpha in primates. Our results demonstrated that TRIM5alpha has been evolving rapidly in primates, which is likely caused by Darwinian positive selection. The SPRY domain of TRIM5alpha, which may be responsible for recognition of incoming viral capsids showed higher nonsynonymous/synonymous substitution ratios than the non-SPRY domain, indicating that the adaptive evolution of TRIM5alpha in primates might be an innate strategy developed in defending retrovirus infection during primate evolution. In addition, the comparative protein sequence analysis suggested that the amino acid substitution pattern at a single site (344R/Q/P) located in the SPRY domain may explain the differences in susceptibilities of HIV-1 infection in diverse primate species.

Accelerated evolution of the pituitary adenylate cyclase-activating polypeptide precursor gene during human origin.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide abundantly expressed in the central nervous system and involved in regulating neurogenesis and neuronal signal transduction. The amino acid sequence of PACAP is extremely conserved across vertebrate species, indicating a strong functional constraint during the course of evolution. However, through comparative sequence analysis, we demonstrated that the PACAP precursor gene underwent an accelerated evolution in the human lineage since the divergence from chimpanzees, and the amino acid substitution rate in humans is at least seven times faster than that in other mammal species resulting from strong Darwinian positive selection. Eleven human-specific amino acid changes were identified in the PACAP precursors, which are conserved from murine to African apes. Protein structural analysis suggested that a putative novel neuropeptide might have originated during human evolution and functioned in the human brain. Our data suggested that the PACAP precursor gene underwent adaptive changes during human origin and may have contributed to the formation of human cognition.