The sex determination cascades in insects are diversified at the top of the cascade, where different primary molecular signals are employed, while at the bottom of the cascades, particularly the doublesex genes, are highly conserved. Here, we identified the doublesex ortholog (Btau-dsx) of Bactrocera tau, a pumpkin fruit fly, and found that Btau-dsx is composed of six exons and five introns with an additional short "m" exon located in the second intron. Btau-dsx is different from its orthologs in most dipteran insects: Its pre-mRNA is sex-specifically spliced to yield three (two male and one female) instead of two transcript variants. The two deduced proteins produced by the male-specific transcripts are a functional (Btau-DSX^M1) and a truncated (Btau-DSX^M2) protein, while the female-specific transcript produces the functional Btau-DSX^F protein. These three proteins contain all conserved domains except Btau-DSX^M2 which has no OD2 domain. The female-specific transcript is detected in both fertilized and unfertilized eggs and in both somatic and germ cells of the adult females, while the male-specific transcript is detected only in fertilized eggs and in the abdominal tissues and testes of adult males. The presence of the Btau-dsx^M1 transcript in fertilized eggs at the early syncytium stage suggests that in XY embryos, the Y-linked M factor gene may function quite soon after fertilization to alter the splicing pattern of Btau-dsx pre-mRNA from the female-specific to the male-specific mode. Injection of Btau-dsx^F dsRNA into recently emerging females can reduce the expression of vitellogenin (Btau-Vg) and causes some defects in the ovaries, indicating that Btau-dsx^F works upstream of Btau-Vg.

Astraeus species are valuable edible ectomycorrhizal mushrooms, particularly in Asia. The partial hypogenous nature of Astraeus fruit bodies in soil present challenges for sample collection and studying species distribution. In this study, we developed a PCR-based approach for identifying Astraeus. Two universal primers (AUPF1/AUPR1 and AUPF3/AUPR3) and specific primers for A. asiaticus (AAF4/ AAR4), A. odoratus (AOF4/AOR4), and A. sirindhorniae (ASF2/ASR4) were designed based on the alignment of internal transcribed spacer sequences from various Astraeus species. Primer verification was performed by generating amplicons from extracted DNA of Astraeus fruit bodies and 130 soil samples collected from beneath various host plants of Astraeus spp. These novel primers were efficient and precise in identifying Astraeus species. Our results have implications for multi-sample assays for Astraeus identification and investigations into species distribution through large-scale inventories.

Astraeus, commonly referred to as the hygroscopic earthstar, barometer earthstar, or false earthstar, is an ectomycorrhizal fungus that is widely consumed in Asia and forms symbiotic associations with tree species belonging to the families Dipterocarpaceae and Pinaceae, among others. This genus is typically distributed in dry dipterocarp forests in northern and northeastern Thailand. However, knowledge on the composition and diversity of fungal communities in areas where Astraeus occurs remains limited. Therefore, this study aimed: (i) to investigate the composition and diversity of soil fungal communities associated with Astraeus across five forest types in Thailand, and (ii) to identify the environmental factors influencing the occurrence of Astraeus species by using NGS (Next-generation sequencing) amplicon sequencing, which targets the Internal Transcribed Spacer 1 (ITS1) region of the rRNA gene. Overall, the results revealed that Astraeus distribution was only in two forest types, namely deciduous dipterocarp forest (DDF) and pine-deciduous dipterocarp forest (PDDF). These forests harbored abundant fungal communities, particularly ectomycorrhizal fungi. Forests where Astraeus was detected (DDF and PDDF) exhibited distinct fungal community composition and diversity compared with forests without Astraeus (dry evergreen forest (DEF), lower montane pine-oak forest (LMPF), and tropical evergreen rain forest (TEF)). Soil properties analysis indicated that Astraeus tends to prefer sandy or sandy loam soils, while showing a negative association with clay soils. These findings emphasize fungal community diversity shape forest ecosystems and the role of the presence or absence of Astraeus. The occurrence of Astraeus is influenced by forest type and soil properties.