Contribution of GPD2/mGPDH to an alternative respiratory chain of the mitochondrial energy metabolism and the stemness in CD133-positive HuH-7 cells.

HuH-7 cells, derived from human hepatocarcinoma, are known to contain the CD133-positive cancer stem cell populations. HuH-7 cells showed higher ATP synthesis activity through the respiratory chain compared to another human hepatocarcinoma cell line HepG2 and showed an especially higher glycerol-3-phosphate (G3P)-driven ATP synthesis (G3P-ATPase) activity. We found that the CD133-positive HuH-7 cells expressed high levels of GPD2 (glycerol-3-phosphate dehydrogenase or mGPDH) and showed high G3P-ATPase activity. Next, to elucidate the relationship between CD133 and GPD2, we inhibited downstream factors of CD133 and found that a p38 inhibitor decreased the expression of GPD2 and decreased the G3P-ATPase activity. Furthermore, GPD2-knockdown (GPD2-KD) cells exhibited strong reduction of the G3P-ATPase activity and reduction of lactic acid secretion. Finally, we validated the effect of GPD2-KD on tumorigenicity. GPD2-KD cells were found to show decreased anchorage-independent cell proliferation, suggesting the linkage of G3P-ATPase activity to the tumorigenicity of the CD133-positive HuH-7 cells. Inhibition of G3P-ATPase disrupts the homeostasis of energy metabolism and blocks cancer development and progression. Our results suggest inhibitors, targeting GPD2 may be potential new anticancer agents.

Involvement of phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin/peroxisome proliferator-activated receptor γ pathway for induction and maintenance of neuropathic pain.

Peripheral nerve injury induces neuropathic pain, which is characterized by the tactile allodynia and thermal hyperalgesia. N-type voltage-dependent Ca2+ channel (VDCC) plays pivotal roles in the development of neuropathic pain, since mice lacking Cav2.2, the pore-forming subunit of N-type VDCC, show greatly reduced symptoms of both tactile allodynia and thermal hyperalgesia. Our study on gene expression profiles of the wild-type and N-type VDCC knockout (KO) spinal cord and several pain-related brain regions after spinal nerve ligation (SNL) injury revealed altered expression of genes encoding catalytic subunits of phosphatidylinositol-3 kinase (PI3K). PI3K/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling is considered to be very important for cancer development and drugs targeting the molecules in this pathway have been tested in oncology trials. In the present study, we have tested whether the changes in expression of molecules in this pathway in mice having spinal nerve injury are causally related to neuropathic pain. Our results suggest that spinal nerve injury induces activation of N-type VDCC and the following Ca2+ entry through this channel may change the expression of genes encoding PI3K catalytic subunits (p110α and p110γ), Akt, retinoid X receptor α (RXRα) and RXRγ. Furthermore, the blockers of the molecules in this pathway are found to be effective in reducing neuropathic pain both at the spinal and at the supraspinal levels. Thus, the activation of PI3K/Akt/mTOR/peroxisome proliferator activated receptor gamma (PPARγ) pathway would be a hallmark of the induction and maintenance of neuropathic pain.

Hydrogen peroxide as a new defensive compound in "benzoyl cyanide" producing polydesmid millipedes.

Hydrogen peroxide was newly and simultaneously demonstrated with well-known hydrogen cyanide as a component of defensive secretions of "benzoyl cyanide" producing polydesmid millipedes. Presence of hydrogen peroxide was successively evidenced by Trinder reagent's spray with colorless as well as oily smears of defensive secretions containing benzoyl cyanide and hydrogen cyanide by alkaline picrate paper treatment. Linear correlation was demonstrated between quantities of hydrogen peroxide and benzoyl cyanide. By qualitative assay, seven benzoyl cyanide containing polydesmidans (six species of adults and one species of a nymph at stadium I) tested positive to Trinder reagent, indicative of the presence of hydrogen peroxide (together with hydrogen cyanide), while two cyanogenic species without benzoyl cyanide exhibited negative responses to the reagent. Two types of millipedes were elucidated as species of cyanogenic Polydesmida.

Chemical polymorphism in defense secretions during ontogenetic development of the millipede Niponia nodulosa.

A mixture of defense compounds (benzaldehyde, benzoyl cyanide, benzoic acid, mandelonitrile, and mandelonitrile benzoate), found commonly in cyanogenic polydesmid millipedes, was identified in the non-cyanogenic millipede Niponia nodulosa. These compounds were major components in 1st-4th instars, but were absent in older instars and adults. Extracts of older instars and adults contained 1-octen-3-ol, 2-methyl-2-bornene, E-2-octen-1-ol, 2-methyl-isoborneol, and geosmin; these compounds were minor components in 1st-4th instars. This ontogenetic allomone shift may be explained by the high cost of biosynthesis of polydesmid compounds from L-phenylalanine being offset by their potency in protecting the insect during fragile and sensitive growth stages. However, as the cuticle hardens in older juveniles (5th, 6th, 7th instars) and adults, this allows for a switch in defense to using less effective and less costly volatile organic compounds (presumably microbial in origin) that are ubiquitous in the millipede's habitat or are produced by symbiotic microbes and may be readily available through food intake or aspiration.

Alleviation of behavioral hypersensitivity in mouse models of inflammatory pain with two structurally different casein kinase 1 (CK1) inhibitors.

BACKGROUND: The phylogenetically highly conserved CK1 protein kinases consisting of at least seven isoforms form a distinct family within the eukaryotic protein kinases. CK1 family members play crucial roles in a wide range of signaling activities. However, the functional role of CK1 in somatosensory pain signaling has not yet been fully understood. The aim of this study was to clarify the role of CK1 in the regulation of inflammatory pain in mouse carrageenan and complete Freund's adjuvant (CFA) models. RESULTS: We have used two structurally different CK1 inhibitors, TG003 and IC261. TG003, which was originally identified as a cdc2-like kinase inhibitor, had potent inhibitory effects on CK1 isoforms in vitro and in cultured cells. Intrathecal injection of either TG003 (1-100 pmol) or IC261 (0.1-1 nmol) dose-dependently decreased mechanical allodynia and thermal hyperalgesia induced by carrageenan or CFA. Bath-application of either TG003 (1 µM) or IC261 (1 µM) had only marginal effects on spontaneous excitatory postsynaptic currents (sEPSCs) recorded in the substantia gelatinosa neurons of control mice. However, both compounds decreased the frequency of sEPSCs in both inflammatory pain models. CONCLUSIONS: These results suggest that CK1 plays an important pathophysiological role in spinal inflammatory pain transmission, and that inhibition of the CK1 activity may provide a novel strategy for the treatment of inflammatory pain.

N-type voltage-dependent Ca2+ channel in non-excitable microglial cells in mice is involved in the pathophysiology of neuropathic pain.

Peripheral nerve injury induces neuropathic pain which is characterized by tactile allodynia and thermal hyperalgesia. N-type voltage-dependent Ca(2+) channel (VDCC) plays pivotal roles in the development of neuropathic pain, since mice lacking Cav2.2, the pore-forming subunit of N-type VDCC, show greatly reduced symptoms of both tactile allodynia and thermal hyperalgesia. Our study on gene expression profiles of the Cav2.2 knockout (KO) spinal cord after spinal nerve ligation (SNL)-injury revealed altered expression of genes known to be expressed in microglia, raising an odd idea that N-type VDCC may function in not only excitable (neurons) but also non-excitable (microglia) cells in neuropathic pain state. In the present study, we have tested this idea by using a transgenic mouse line, in which suppression of Cav2.2 expression can be achieved specifically in microglia/macrophage by the application of tamoxifen. We found SNL-operated transgenic mice exhibited greatly reduced signs of tactile allodynia, whereas the degree of thermal hyperalgesia was almost the same as that of control. Immunohistochemical analysis of the transgenic lumbar spinal cord revealed reduced accumulation of Iba1-positive cells (microglia/macrophage) around the injured neurons, indicating microglial N-type VDCC is important for accumulation of microglia at the lesion sites. Although the mechanism of its activation is not clear at present, activation of N-type VDCC expressed in non-excitable microglial cells contributes to the pathophysiology of neuropathic pain.

GPD2: The relationship with cancer and neural stemness.

We previously reported that knocking down GPD2 (glycerol-3-phosphate dehydrogenase 2), responsible for the glycerol-phosphate shuttle, causes human hepatocarcinoma-derived HuH-7 cells, lowering the cancer stemness. After examining whether GPD2 expression in the other cell lines could affect their cancer stemness, this study showed that human neuroblastoma-derived SH-SY5Y cells also lower the ability of sphere formation by knocking down GPD2. This suggests that GPD2 relates to the common mechanism for maintaining cancer stem cells, as in the cases like SH-SY5Y and HuH-7 cells. In addition, knocking down GPD2 in SH-SY5Y cells showed a morphological change and increasing tendency of neuronal marker genes, including GAP43, NeuN, and TUBB3, indicating that GPD2 may contribute to not only cancer but also neural stem cell maintenance. After all, GPD2 may play a role in maintaining cancer and neural stemness, although further rigorous studies are essential to conclude this. It is expected that GPD2 will be a novel target gene for cancer therapy, stem cell research, and development.

n-Hexyl laurate and fourteen related fatty acid esters: new secretory compounds from the julid millipede, Anaulaciulus sp.

A total of fifteen saturated fatty acid esters were newly identified from the secretions of an unidentified Anaulaciulus sp. (Julida: Julidae). The fatty acid components of the esters were composed of normal chain acids (from C(10) to C(14)) and of branched chain acids (from iso-C(12) to iso-C(15) and anteiso-C(15)). The alcohol moieties were all composed of normal chain alcohols varying from n-butanol to n-octanol. The most abundant component found in the total esters was n-hexyl laurate (64.7%). Novel compounds identified from the millipede secretion extracts include six branched iso- and anteiso-fatty esters, an odd-numbered C(11)-fatty acid ester, a C(13)-fatty acid ester, and a C(7)-alcohol ester, all of which were previously undescribed natural products. In addition, a characteristic mixture of benzoquinones, such as 2-methyl-1,4-benzoquinone, 2-methoxy-3-methyl-1,4-benzoquinone, 2,3-dimethoxy-1,4-benzoquinone, 2-methoxy-6-methyl-1,4-benzoquinone, and 2,3-dimethoxy-5-methyl-1,4-benzoquinone were identified from the secretions, together with trace amounts of 1,4-benzoquinone.

Release of hydrogen cyanide via a post-secretion Schotten-Baumann reaction in defensive fluids of polydesmoid millipedes.

Mandelonitrile benzoate, a minor defense component produced by polydesmoid millipedes, is produced in large amounts together with hydrogen cyanide following shake-disturbances administered to individuals of Nedyopus tambanus tambanus, Parafontaria tonominea, Epanerchodus sp., and Epanerchodus fulvus. These species commonly produce mandelonitrile and benzoyl cyanide (the oxidized product after discharge). The newly generated mandelonitrile benzoate was identified as a product of post secretion Schotten-Baumann reaction under basic conditions of bled bodily fluids (pH ca. 9.0), and was not an enzymatic reaction product. The reaction occurred in vitro even under less basic conditions [1M Tris-HCl buffer (pH 8.0)], and could be defined as a new mechanism of hydrogen cyanide release occurring in roughly half of polydesmoid millipedes. Species possessing no benzoyl cyanide, such as Oxidus gracilis and Cryptocorypha sp., could also produce mandelonitrile benzoate under conditions in which benzoyl cyanide was exogenously provided.

Symbiosis of the millipede parasitic nematodes Rhigonematoidea and Thelastomatoidea with evolutionary different origins.

BACKGROUND: How various host-parasite combinations have been established is an important question in evolutionary biology. We have previously described two nematode species, Rhigonema naylae and Travassosinema claudiae, which are parasites of the xystodesmid millipede Parafontaria laminata in Aichi Prefecture, Japan. Rhigonema naylae belongs to the superfamily Rhigonematoidea, which exclusively consists of parasites of millipedes. T. claudiae belongs to the superfamily Thelastomatoidea, which includes a wide variety of species that parasitize many invertebrates. These nematodes were isolated together with a high prevalence; however, the phylogenetic, evolutionary, and ecological relationships between these two parasitic nematodes and between hosts and parasites are not well known. RESULTS: We collected nine species (11 isolates) of xystodesmid millipedes from seven locations in Japan, and found that all species were co-infected with the parasitic nematodes Rhigonematoidea spp. and Thelastomatoidea spp. We found that the infection prevalence and population densities of Rhigonematoidea spp. were higher than those of Thelastomatoidea spp. However, the population densities of Rhigonematoidea spp. were not negatively affected by co-infection with Thelastomatoidea spp., suggesting that these parasites are not competitive. We also found a positive correlation between the prevalence of parasitic nematodes and host body size. In Rhigonematoidea spp., combinations of parasitic nematode groups and host genera seem to be fixed, suggesting the evolution of a more specialized interaction between Rhigonematoidea spp. and their host. On the other hand, host preference of Thelastomatoidea spp. was not specific to any millipede species, indicating a non-intimate interaction between these parasites and their hosts. CONCLUSIONS: The two nematode superfamilies, Rhigonematoidea and Thelastomatoidea, have phylogenetically distinct origins, and might have acquired xystodesmid millipede parasitism independently. Currently, the two nematodes co-parasitize millipedes without any clear negative impact on each other or the host millipedes. Our study provides an example of balanced complex symbioses among parasitic nematodes and between parasitic nematodes and host millipedes, which have been established over a long evolutionary history.

Genetic recombination in disgust-associated bitter taste-responsive neurons of the central nucleus of amygdala in male mice.

A bitter substance induces specific orofacial and somatic behavioral reactions such as gapes in mice as well as monkeys and humans. These reactions have been proposed to represent affective disgust, and therefore, understanding the neuronal basis of the reactions would pave the way to understand affective disgust. It is crucial to identify and access the specific neuronal ensembles that are activated by bitter substances, such as quinine, the intake of which induces disgust reactions. However, the method to access the quinine-activated neurons has not been fully established yet. Here, we show evidence that a targeted recombination in active populations (TRAP) method, induces genetic recombination in the quinine-activated neurons in the central nucleus of the amygdala (CeA). CeA is one of the well-known emotional centers of the brain. We found that the intraoral quinine infusion, that resulted in disgust reactions, increased both cFos-positive cells and Arc-positive cells in the CeA. By using Arc-CreER;Ai3 TRAP mice, we induced genetic recombination in the quinine-activated neurons and labelled them with fluorescent protein. We confirmed that the quinine-TRAPed fluorescently-labelled cells preferentially coexpressed Arc after quinine infusion. Our results suggest that the TRAP method can be used to access specific functional neurons in the CeA.

Multiple speciation events in an arthropod with divergent evolution in sexual morphology.

Sexual selection can facilitate divergent evolution of traits related to mating and consequently promote speciation. Theoretically, independent operation of sexual selection in different populations can lead to divergence of sexual traits among populations and result in allopatric speciation. Here, we show that divergent evolution in sexual morphology affecting mating compatibility (body size and genital morphologies) and speciation have occurred in a lineage of millipedes, the Parafontaria tonominea species complex. In this millipede group, male and female body and genital sizes exhibit marked, correlated divergence among populations, and the diverged morphologies result in mechanical reproductive isolation between sympatric species. The morphological divergence occurred among populations independently and without any correlation with climatic variables, although matching between sexes has been maintained, suggesting that morphological divergence was not a by-product of climatic adaptation. The diverged populations underwent restricted dispersal and secondary contact without hybridization. The extent of morphological difference between sympatric species is variable, as is diversity among allopatric populations; consequently, the species complex appears to contain many species. This millipede case suggests that sexual selection does contribute to species richness via morphological diversification when a lineage of organisms consists of highly divided populations owing to limited dispersal.

Deficit of heat shock transcription factor 1-heat shock 70 kDa protein 1A axis determines the cell death vulnerability in a model of spinocerebellar ataxia type 6.

Spinocerebellar ataxia type 6 (SCA6) is caused by a small expansion of polyglutamine (polyQ)-encoding CAG repeat in Ca(v)2.1 calcium channel gene. To gain insights into pathogenic mechanism of SCA6, we used HEK293 cells expressing fusion protein of enhanced green fluorescent protein and Ca(v)2.1 carboxyl terminal fragment (EGFP-Ca(v)2.1CT) [L24 and S13 cells containing 24 polyQ (disease range) and 13 polyQ (normal range), respectively] and examined their responses to some stressors. When exposed to CdCl(2), L24 cells showed lower viability than the control S13 cells and caspase-dependent apoptosis was enhanced more in L24 cells. Localization of EGFP-Ca(v)2.1CT was almost confined to the nucleus, where it existed as speckle-like structures. Interestingly, CdCl(2) treatment resulted in disruption of more promyelocytic leukemia nuclear bodies (PML-NBs) in L24 cells than in S13 cells and in cells where PML-NBs were disrupted, aggregates of EGFP-Ca(v)2.1CT became larger. Furthermore, a large number of aggregates were formed in L24 cells than in S13 cells. Results of RNAi experiments indicated that HSPA1A determined the difference against CdCl(2) toxicity. Furthermore, protein expression of heat shock transcription factor 1 (HSF1), which activates HSPA1A expression, was down-regulated in L24 cells. Therefore, HSF1-HSPA1A axis is critical for the vulnerability in L24 cells.

Knockdown of microglial Cav2.2 N-type voltage-dependent Ca2+ channel ameliorates behavioral deficits in a mouse model of Parkinson's disease.

Cav2.2 N-type voltage-dependent Ca2+ channel (VDCC) expressed in neurons is known to be essential for neurotransmitter release. We have shown previously that this channel is also expressed in nonexcitable microglia and plays pivotal roles in microglial functions. Here, we have examined the effects of microglia-specific knockdown (KD) of Cav2.2 channel in a mouse model of Parkinson's disease (PD). We found that the KD of Cav2.2 channel reduces the accumulation of microglia in the substantia nigra and ameliorates the behavioral deficits in PD model mice. These results are in marked contrast with those found in microglia-specific KD of Cav1.2 L-type channel, where exacerbated symptoms are observed. Our results suggest that blockade of microglial Cav2.2 N-type VDCC is beneficial for the treatment of PD.

Upregulation of casein kinase 1epsilon in dorsal root ganglia and spinal cord after mouse spinal nerve injury contributes to neuropathic pain.

BACKGROUND: Neuropathic pain is a complex chronic pain generated by damage to, or pathological changes in the somatosensory nervous system. Characteristic features of neuropathic pain are allodynia, hyperalgesia and spontaneous pain. Such abnormalities associated with neuropathic pain state remain to be a significant clinical problem. However, the neuronal mechanisms underlying the pathogenesis of neuropathic pain are complex and still poorly understood. Casein kinase 1 is a serine/threonine protein kinase and has been implicated in a wide range of signaling activities such as cell differentiation, proliferation, apoptosis, circadian rhythms and membrane transport. In mammals, the CK1 family consists of seven members (alpha, beta, gamma1, gamma2, gamma3, delta, and epsilon) with a highly conserved kinase domain and divergent amino- and carboxy-termini. RESULTS: Preliminary cDNA microarray analysis revealed that the expression of the casein kinase 1 epsilon (CK1epsilon) mRNA in the spinal cord of the neuropathic pain-resistant N- type Ca2+ channel deficient (Cav2.2-/-) mice was decreased by the spinal nerve injury. The same injury exerted no effects on the expression of CK1epsilon mRNA in the wild-type mice. Western blot analysis of the spinal cord identified the downregulation of CK1epsilon protein in the injured Cav2.2-/- mice, which is consistent with the data of microarray analysis. However, the expression of CK1epsilon protein was found to be up-regulated in the spinal cord of injured wild-type mice. Immunocytochemical analysis revealed that the spinal nerve injury changed the expression profiles of CK1epsilon protein in the dorsal root ganglion (DRG) and the spinal cord neurons. Both the percentage of CK1epsilon-positive neurons and the expression level of CK1epsilon protein were increased in DRG and the spinal cord of the neuropathic mice. These changes were reversed in the spinal cord of the injured Cav2.2-/- mice. Furthermore, intrathecal administration of a CK1 inhibitor IC261 produced marked anti-allodynic and anti-hyperalgesic effects on the neuropathic mice. In addition, primary afferent fiber-evoked spinal excitatory responses in the neuropathic mice were reduced by IC261. CONCLUSIONS: These results suggest that CK1epsilon plays important physiological roles in neuropathic pain signaling. Therefore CK1epsilon is a useful target for analgesic drug development.

Involvement of N-type Ca2+ channel in microglial activation and its implications to aging-induced exaggerated cytokine response.

Voltage-dependent calcium channel (VDCC) is generally believed to be active only in excitable cells. However, we have reported recently that N-type VDCC (Cav2.2) could become functional in non-excitable cells under pathological conditions. In the present study, we show that Cav2.2 channels are also functional in physiological microglial activation process. By using a mouse microglial cell line (MG6), we examined the effects of a Cav2.2 blocker on the activation of MG6 cells, when treated with lipopolysaccharide (LPS) / interferon γ (IFNγ) or with interleukin-4 (IL-4). As a result, blocking the activation of Cav2.2 enhanced so-called alternative activation process of microglia (transition to neuroprotective M2 microglia) without changing the efficacy of the transition to neuroinflammatory M1 microglia. This enhanced M2 transition involved the activation of a transcription factor hypoxia inducible factor 2 (HIF-2), since a specific blocker of HIF-2 completely abolished this enhancement. We then examined whether Cav2.2 activation was involved in aging-related neuroinflammation. Using primary culture of microglia, we found that the efficacy of microglial M1 transition was enhanced but that M2 transition was reduced by aging, in agreement with a general notion that aging induces enhanced neuroinflammation. Finally, we show here that the moderate blockade of Cav2.2 expression in microglia restores this age-dependent reduction of microglial M2 transition and reduces the aging-induced exaggerated cytokine response, as revealed by a fast recovery from depressive-like behaviors in microglia-specific Cav2.2 deficient mice. These results suggest a critical role for microglial Cav2.2 channel in the aging-related neuroinflammation.

Genetic Access to Gustatory Disgust-Associated Neurons in the Interstitial Nucleus of the Posterior Limb of the Anterior Commissure in Male Mice.

Orofacial and somatic disgust reactions are observed in rats following intraoral infusion of not only bitter quinine (innate disgust) but also sweet saccharin previously paired with illness (learned disgust). It remains unclear, however, whether these innate and learned disgust reactions share a common neural basis and which brain regions, if any, host it. In addition, there is no established method to genetically access neurons whose firing is associated with disgust (disgust-associated neurons). Here, we examined the expression of cFos and Arc, two markers of neuronal activity, in the interstitial nucleus of the posterior limb of the anterior commissure (IPAC) of male mice that showed innate disgust and mice that showed learned disgust. Furthermore, we used a targeted recombination in active populations (TRAP) method to genetically label the disgust-associated neurons in the IPAC with YFP. We found a significant increase of both cFos-positive neurons and Arc-positive neurons in the IPAC of mice that showed innate disgust and mice that showed learned disgust. In addition, TRAP following quinine infusion (Quinine-TRAP) resulted in significantly more YFP-positive neurons in the IPAC, compared to TRAP following water infusion. A significant number of the YFP-positive neurons following Quinine-TRAP were co-labeled with Arc following the second quinine infusion, confirming that Quinine-TRAP preferentially labeled quinine-activated neurons in the IPAC. Our results suggest that the IPAC activity is associated with both innate and learned disgust and that disgust-associated neurons in the IPAC are genetically accessible by TRAP.

Blockade of microglial Cav1.2 Ca2+ channel exacerbates the symptoms in a Parkinson's disease model.

Cav1.2 channels are an L-type voltage-dependent Ca2+ channel, which is specifically blocked by calcium antagonists. Voltage-dependent Ca2+ channels are generally considered to be functional only in excitable cells like neurons and muscle cells, but recently they have been reported to also be functional in non-excitable cells like microglia, which are key players in the innate immune system and have been shown to be involved in the pathophysiology of Parkinson's disease. Here, we show that Cav1.2 channels are expressed in microglia, and that calcium antagonists enhanced the neuroinflammatory M1 transition and inhibited neuroprotective M2 transition of microglia in vitro. Moreover, intensive degeneration of dopaminergic neurons and accompanying behavioural deficits were observed in microglia-specific Cav1.2 knockdown mice intoxicated with MPTP, a neurotoxin that induces Parkinson's disease-like symptoms, suggesting detrimental effects of microglial Cav1.2 blockade on Parkinson's disease. Therefore, microglial Cav1.2 channel may have neuroprotective roles under physiological conditions and may also contribute to recovery from disease conditions.

Complex copulatory behavior and the proximate effect of genital and body size differences on mechanical reproductive isolation in the millipede genus Parafontaria.

The role of species-specific genitalia in reproductive isolation is unclear. Males of the millipede genus Parafontaria use gonopods (modified eighth legs) charged with sperm from the genital openings of the second legs as intromittent organs. Males perform both preliminary and true intromission during mating. During preliminary intromission, a male attempts to insert his gonopods into the female genitalia before charging the gonopods with sperm. If this intromission is completed, it is followed by the ejaculation of sperm to the gonopods and true intromission for insemination. In two sympatric species of Parafontaria that lack effective precopulatory isolation, copulation was terminated without insemination because of preliminary intromission failure caused by mismatched genital and body sizes. Thus, mechanical isolation between these sympatric species resulted from morphological differentiation mediated by the obligatory preliminary intromission. These findings demonstrate the proximate importance of genital and body size differences for reproductive isolation within this genus of millipede.

Biosynthesis of (2-nitroethyl)benzene and (Z)- and (E)-(2-nitroethenyl)benzenes from (Z)- and (E)-phenylacetaldoximes and phenylacetonitrile; defense allomone of Eutrichodesmus elegans and Eutrichodesmus armatus (Polydesmida: Haplodesmidae).

The defense allomones of two haplodesmid millipedes, Eutrichodesmus elegans and E. armatus (Polydesmida: Haplodesmidae), are known as a mixture of the following three nitro compounds: (2-nitroethyl)benzene and (Z)- and (E)-(2-nitroethenyl)benzenes. Administrations of a mixture of 2H-labeled (Z)- and (E)-phenylacetaldoximes and of 2H-labeled phenylacetonitrile as precursors resulted in the same production of three 2H-labeled nitro compounds, [2'-nitroethyl][2,3,4,5,6-2H5]benzene and [(Z)- and (E)-2'-nitroethenyl][2,3,4,5,6-2H5]benzenes, in both species. Oxime administration at an appropriate dose resulted in the production of three nitro compounds with similar natural ratios more effectively than nitrile administration. Conversion from oximes to nitrile and vice versa was evidenced during administration. Occurrences of three precursors (Z- and E-oximes and nitrile) were detected sporadically in millipede extracts by selected ion chromatography.