Circulating Cell-Free DNA Profiling Predicts the Therapeutic Outcome in Advanced Hepatocellular Carcinoma Patients Treated with Combination Immunotherapy.

Combination immunotherapy with anti-programmed cell death1-ligand1 (PD-L1) and anti-vascular endothelial growth factor (VEGF) antibodies has become the standard treatment for patients with unresectable HCC (u-HCC). However, limited patients obtain clinical benefits. Cell-free DNA (cfDNA) in peripheral blood contains circulating tumor DNA (ctDNA) that reflects molecular abnormalities in tumor tissue. We investigated the potential of cfDNA/ctDNA as biomarkers for predicting the therapeutic outcome in u-HCC patients treated with anti-PD-L1/VEGF therapy. We enrolled a multicenter cohort of 85 HCC patients treated with atezolizumab and bevacizumab (Atezo/Bev) between 2020 and 2021. Pretreatment plasma was collected, and cfDNA levels were quantified. Ultradeep sequencing of cfDNA was performed with a custom-made panel for detecting mutations in 25 HCC-related cancer genes. We evaluated the association of cfDNA/ctDNA profiles and clinical outcomes. Patients with high plasma cfDNA levels showed a significantly lower response rate and shorter progression-free survival and overall survival (OS) than those with low cfDNA levels. ctDNA detected in 55% of HCC patients included the telomerase reverse transcriptase (TERT) promoter in 31% of these patients, tumor protein 53 (TP53) in 21%, catenin beta 1 (CTNNB1) in 13% and phosphatase and tensin homolog (PTEN) in 7%. The presence or absence of ctDNA did not predict the efficacy of Atezo/Bev therapy. Twenty-six patients with a TERT mutation had significantly shorter OS than those without. The presence of a TERT mutation and alpha-fetoprotein (AFP) ≥ 400 ng/mL were independent predictors of poor OS according to multivariate Cox proportional hazard analysis and could be used to stratify patients treated with Atezo/Bev therapy based on prognosis. In conclusion, pretreatment cfDNA/ctDNA profiling may be useful for predicting the therapeutic outcome in u-HCC patients treated with anti-PD-L1/VEGF therapy.

No selection for change in polyandry under experimental evolution.

What drives mating system variation is a major question in evolutionary biology. Female multiple mating (polyandry) has diverse evolutionary consequences, and there are many potential benefits and costs of polyandry. However, our understanding of its evolution is biased towards studies enforcing monandry in polyandrous species. What drives and maintains variation in polyandry between individuals, genotypes, populations and species remains poorly understood. Genetic variation in polyandry may be actively maintained by selection, or arise by chance if polyandry is selectively neutral. In Drosophila pseudoobscura, there is genetic variation in polyandry between and within populations. We used isofemale lines to found replicate populations with high or low initial levels of polyandry and tracked polyandry under experimental evolution over seven generations. Polyandry remained relatively stable, reflecting the starting frequencies of the experimental populations. There were no clear fitness differences between high versus low polyandry genotypes, and there was no signature of balancing selection. We confirmed these patterns in direct comparisons between evolved and ancestral females and found no consequences of polyandry for female fecundity. The absence of differential selection even when initiating populations with major differences in polyandry casts some doubt on the importance of polyandry for female fitness.

Male behavioural plasticity depends on maternal mating status in the two-spotted spider mite.

In haplodiploid organisms including the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae), both unmated and mated females can produce male offspring. A previous study reported that males produced by unmated females (UM males) find pre-reproductive females more quickly than males produced by mated females (M males) in T. urticae. However, it remains unclear what factors cause the difference. We investigated effects of maternal mating status on mate-searching behaviour of their sons by changing the sons' developmental environment. In T. urticae, the primary sex ratio of mated-female colonies is female-biased. For both UM and M males, half of individuals were reared with males to imitate unmated-female colonies, whereas the rest were reared with females to imitate mated-female colonies. In UM males, individuals that had developed with males found pre-reproductive females more quickly than those that had developed with females. However, such a difference was not observed in M males. This indicates that behavioural response to the developmental environment differs between UM and M males. It means that the behavioural plasticity depends on maternal mating status. When males were individually reared, however, there was no significant difference in the mate-searching behaviour between UM and M males, indicating that maternal mating status does not independently affect their sons' mate-searching behaviour. This study showed that male mate-searching behaviour is changed by their developmental environment and maternal mating status. This behavioural plasticity depending on maternal mating status is the first reported in haplodiploid organisms.

Precopulatory mate guarding influences the development of quiescent deutonymph females in the two-spotted spider mite (Acari: Tetranychidae).

Individuals of some organisms have a specific stage sensitive to environmental cues that initiate developmental plasticity which subsequently influences their entire development. Females may use male behaviour such as precopulatory mate guarding as an environmental cue to change their developmental rate. In the two-spotted spider mite Tetranychus urticae (Acari: Tetranychidae), only the first insemination results in fertilization and males guard quiescent deutonymph females. As quiescent individuals take on a silvery appearance before moulting, the period of the quiescent stage can be divided into two parts: from entering the quiescent stage to becoming silvery (1st period) and from becoming silvery to moulting (2nd period). Females may be sensitive to precopulatory mate guarding immediately before moulting (i.e. 2nd period). Thus, I examined whether precopulatory mate guarding during either period affects the total developmental duration of quiescent deutonymph females. When guarded by a male, the whole developmental duration of the quiescent deutonymph females became significantly shorter (by 3-5%) than that of solitary ones, regardless whether the guarding occurred during the 1st period, the 2nd period or both periods. In conclusion, quiescent deutonymph T. urticae females use precopulatory mate guarding by conspecific males as an environmental cue for their developmental rate, although they are sensitive to the mate guarding not only immediately before moulting.

Altered volatile profile associated with precopulatory mate guarding attracts spider mite males.

Proximate factors affecting animal behavior include stimuli generated by conspecifics. In spider mites of the genus Tetranychus (Acari: Tetranychidae), males guard pre-reproductive quiescent females, because only the first mating results in fertilization. In a dual-choice experiment, more adult males of T. urticae were attracted to females guarded by a male than to solitary females. Because spider mites are known to perceive volatiles, we hypothesized that guarded and solitary females differ in the volatile blends emitted. To test this hypothesis, headspace volatiles of guarded females, solitary females, and solitary males were collected, respectively. GC/MS analysis detected octanal, methyl salicylate, ethyl 4-ethoxybenzoate, and methyl cis-dihydrojasmonate in all of the groups. Orthogonal Projection to Latent Structures Discriminant Analysis (OPLS-DA) of the blends clearly discriminated guarded females from solitary females, supporting our hypothesis. Individual compounds did not show significant difference in emission rates for guarded females vs. solitary females, suggesting that differences lay in the total blend composition. OPLS-DA did not discriminate between the blends emitted by guarded females and solitary males. In conclusion, the differences in the volatile blends are likely to mediate male discrimination between guarded and solitary females.

Indirect evidence that guarded quiescent deutonymph females invest energy to attract conspecific males in the Kanzawa spider mite (Acari: Tetranychidae)?

Because only the first mating results in fertilization in Tetranychus kanzawai (Acari: Tetranychidae), adult males guard quiescent deutonymph females (i.e., precopulatory mate guarding). A previous study reported that quiescent deutonymph females guarded by a male attract more conspecific males than solitary females and then hypothesized that guarded females release more chemical signals than solitary ones to attract males. Quiescent deutonymph females do not feed. If the hypothesis is appropriate, guarded females should invest energy in attracting males at the expense of investment in other activities, such as egg production. Therefore, we compared oviposition rates immediately after adult emergence between guarded females and solitary females. On the first day, the oviposition rate of guarded females was lower than that of solitary females. On the second day, however, there was no significant difference between female groups. These results suggest that guarded females invest energy in activities other than egg production before adult emergence and that the energetic cost is easily recoverable. We believe that our finding indirectly supports the hypothesis that guarded females release more chemical signals than solitary females to attract conspecific males.

Effects of age and mating on female sex attractant pheromone levels in the sorghum plant bug, Stenotus rubrovittatus (Matsumura).

Previous work has shown that male sorghum plant bugs, Stenotus rubrovittatus (Matsumura) (Heteroptera: Miridae), are more attracted to young virgin females than to old virgin females and are not attracted to mated females. Therefore, we examined the effects of age and mating status on female sex pheromone levels. The pheromone components, hexyl butyrate, (E)-hex-2-en-1-yl butyrate, and (E)-4-oxohex-2-enal, were collected from females in two different ways. First, the compounds were extracted from whole bodies of each female and quantified by gas chromatography-mass spectrometry (GC-MS). Pheromone levels extracted from virgin female bodies decreased with age. In females that had just mated, pheromone levels did not differ from those of virgin females, and pheromone levels in mated females remained relatively constant up to 15 d after mating. This absence of change in pheromone levels extracted from whole bodies of mated females is not congruent with previous reports of lack of male attraction to these females. In a second method for pheromone recovery (adsorption on a PDMS-coated stir bar), GC-MS analysis showed that mated females released lower amounts of pheromone components compared to those emitted by virgin females of the same age (4 d). As was the case with whole body extracts, young virgin females (3 d) released higher amounts of pheromone components than did old virgin females (8 d). The results suggest that male response in S. rubrovittatus is dictated more by the quantities of pheromone components released into the volatile headspace by females than by the quantities present in the body.

Males of the two-spotted spider mite attempt to copulate with mated females: effects of double mating on fitness of either sex.

In Tetranychus urticae (Acari: Tetranychidae), when the intervals between first and second copulation are more than 24 h, only the first copulation is effective for females. Therefore, adult males should copulate only with virgin females, but not with females that copulated more than 1 day ago. Indeed, T. urticae males preferred virgin females to mated females under dual choice conditions. In the absence of virgin females, however, 60% of males copulated with mated females (n = 30). Therefore, the effects of male copulation behaviour on male and mated-female fitness were examined, respectively. Since T. urticae is arrhenotokous (i.e., only daughters have genes derived from their father), the proportion of females among the offspring was used as an index of male fitness. After males had lived with/without a mated female, the males were allowed to copulate with a virgin female. The proportion of females among the offspring did not differ between males with and without a female. On the other hand, when mated females lived with an adult male, their egg production was lower than mated females without a male. These results suggest that males do not seem to obtain fitness benefit from the copulation behaviour and that mated females incur a fitness cost due to the male behaviour.

The presence of webbing affects the oviposition rate of two-spotted spider mites, Tetranychus urticae (Acari: Tetranychidae).

Several species of tetranychid mites including Tetranychus urticae Koch (Acari: Tetranychidae) construct complicated three-dimensional webs on plant leaves. These webs provide protection against biotic and abiotic stress. As producing web is likely to entail a cost, mites that arrive on a leaf with web are expected to refrain from producing it, because they will gain the benefit of protection from the existing web. Mites that produce less web may then allocate resources that are not spent on web construction to other fitness-enhancing activities, such as laying eggs. To test this, the oviposition rate of T. urticae adult females was examined on leaves with web. As a control, we used leaves where the web had been removed, hence both types of leaves had been exposed to conspecifics previously and were thus damaged. On leaves with web, the oviposition rate of T. urticae females was higher than on leaves where the web had been removed. Therefore, the presence of web constructed by conspecifics enhanced the oviposition rate of T. urticae females. This provides indirect evidence that mites use the web constructed by conspecifics and thereby save resources that can be allocated to other traits that enhance reproductive success.

Genetic differentiation between resistance phenotypes in the phytophagous flea beetle, Phyllotreta nemorum.

The flea beetle Phyllotreta nemorum L. (Coleoptera: Chrysomelidae) is genetically polymorphic for resistance against the defences of one of its host plants, Barbarea vulgaris R.Br. (Brassicales: Brassicaceae). Whereas resistant flea beetles are able to use B. vulgaris as well as other cruciferous plants as food, non-resistant beetles cannot survive on B. vulgaris. This limitation to host plant use of non-resistant beetles could potentially lead to asymmetric gene flow and some degree of genetic isolation between the different resistance-genotypes. Therefore, we studied the extent of genetic differentiation at neutral allozyme loci between samples of flea beetles that were collected at different locations and first tested for resistance phenotype. Since earlier work has shown a weak, but significant, effect of geographical distance between the samples on their genetic differentiation, in the present study variation at the neutral allozyme loci in P. nemorum was partitioned between geographical distance and resistance-phenotype. Both sources independently contributed statistically significantly to population differentiation. Thus, there appears to be a limitation to genetic exchange between the resistant and non-resistant flea beetles when corrections are made for their geographic differentiation. This is consistent with the presence of some degree of host race formation in this flea beetle.

Is only the first mating effective for females in the Kanzawa spider mite, Tetranychus kanzawai (Acari: Tetranychidae)?

Although only the first mating is effective for females in Tetranychus urticae (Acari: Tetranychidae), it remains unclear whether this is also true for closely related species, such as T. kanzawai. To address this question, I analyzed paternity in the progeny of T. kanzawai females that had been observed to copulate with two males by using a microsatellite DNA marker. In this study, mating was allowed to take place without experimental interruption. The results show that progenies were sired by both males in only 1 of 14 families, whereas progeny were sired only by the first males in the other families. This result suggests that only the first mating would be, by and large, effective in T. kanzawai.

Spider mites assess predation risk by using the odor of injured conspecifics.

When a predatory mite, Amblyseius womersleyi is present, Tetranychus kanzawai takes refuge on its webs during a quiescent stage. To examine the factors responsible for the antipredator behavior of T. kanzawai, we exposed T. kanzawai to the odor of conspecifics, A. womersleyi, and both T. kanzawai and A. womersleyi. The proportion of T. kanzawai females that took refuge was significantly higher when they were exposed to the odor of 'both T. kanzawai and A. womersleyi.' Furthermore, the proportion of T. kanzawai females that took refuge was significantly higher when they were exposed to injured conspecifics than to intact conspecifics. These results suggest that T. kanzawai assesses predation risk, at least in part, by using the odor of injured conspecifics.