Inferring the Origin of <i>Cylas formicarius</i>（Coleoptera: Brentidae）Found and Eradicated in Hamamatsu City, Shizuoka Prefecture, Using Molecular Phylogenetic Analysis

Efforts to reduce smoking at the Olympics that will be hosted in Tokyo in 2020 are spreading across the country, but they are not enough. In general, it is said that (1) smoke prevention education for minors, (2) smoking cessation support for smokers, and (3) smoking restriction in the social environment are necessary to reduce the smoking rate. Healthy Japan 21 called for achieving a numerical target for the reduction of youth smoking by 2010. The author is a principal school doctor at Hamamatsu Municipal Yuto Elementary School and has been teaching antismoking classes to elementary school students since 1999. In this area (Yuto District, Hamamatsu City in Shizuoka Prefecture, Japan), there is the Oki-jinja Shrine, which was built in 705, and a large event (Oki-jinja grand festival) is held there each autumn, attracting 2500 people. Since many people gather there, it is worthwhile to provide information about passive smoking prevention on shrine grounds. Since 1999, smoking prevention classes have been held at elementary schools, and in 2008, smoking was prohibited in the grounds of the Oki-jinja Shrine, making the festival there totally nonsmoking within the shrine grounds, among additional measures taken against smoking. In addition, measures against smoking at large events in Shizuoka Prefecture and Hamamatsu City have also been effective, and the smoking rate among Hamamatsu citizens has dropped to 10.2% (From Health Promotion Division, Hamamatsu City, 2016), which is expected to extend their healthy life expectancy. To reduce the smoking rate, it is important not only to provide administrative support but also to engage in steady civic activities.

To extract factors for the spread and continuation of forest certification, it is important to conduct comparative studies among certified business entities and understand the expectations and perceived effects of certification on the entire supply chain. This study classified industries into forestry (timber production), sawmills (processing), and construction (consumption), and then analyzed the expectations and perceived effects of forest certification. Data for analysis were taken from questionnaire and interview surveys among certified business entities in Hamamatsu City, Shizuoka Prefecture, which has the largest area covered by Forest Stewardship Council certification in Japan. Social benefits were cited more often than economic benefits as incentives for obtaining certification. Analysis of the difference between expectations and awareness of the effects revealed that satisfaction was the lowest in the sawmill entities. The sawmill entities received less administrative support compared with the other two business types and tended to be dependent on trends obtaining certification in forestry and construction entities. If sawmill entities do not find forest certification appealing, and the number of business entities obtaining forest certification declines, then the supply chain for certified timber will no longer function. The government must increase awareness of the importance of linking forest certification throughout the supply chain and implement forest certification support projects evenly across all business types.

Biennial bearing, the biggest factor destabilizing citrus fruit production, is primarily attributed to unfavorable weather changes. However, the relationship between the yield of Satsuma mandarin for more than 50 years and the weather remains poorly understood. Therefore, the present study investigated the effect of meteorological factors, such as average temperature, precipitation, and sunshine duration, on the yield of Satsuma mandarin in Shizuoka Prefecture, Japan. We analyzed fruit production statistics and meteorological observation data from 1961 to 2018. Furthermore, we investigated the alternative indices of flowering and sprouting in each of the 20 districts of Mikkabi Town, Hamamatsu City, Shizuoka Prefecture. We detected a positive correlation between the average temperatures in March and February and yields in Shizuoka and Hamamatsu Cities. All alternative indices, except flowering in the early-maturing cultivar, were significantly lower in the southern than in the northern districts (P < 0.01 or 0.05). Particularly, the alternative index of flowering in the late-maturing cultivar was significantly different between the northern and southern districts (P < 0.01). Furthermore, we found a negative correlation between the mean annual temperature or average temperature from January to April and alternative indices of flowering and sprouting. The findings of our field survey in Mikkabi production area suggest that mean annual temperature may affect the biennial bearing of Satsuma mandarin. © 2023 Society of Chemical Industry.

The fruit fly Bactrocera caudata is a pest species of economic importance in Asia. Its larvae feed on the flowers of Cucurbitaceae such as Cucurbita moschata. To-date it is distinguished from related species based on morphological characters. Specimens of B. caudata from Peninsular Malaysia and Indonesia (Bali and Lombok) were analysed using the partial DNA sequences of cytochrome c oxidase subunit I (COI) and 16S rRNA genes. Both gene sequences revealed that B. caudata from Peninsular Malaysia was distinctly different from B. caudata of Bali and Lombok, without common haplotype between them. Phylogenetic analysis revealed two distinct clades, indicating distinct genetic lineage. The uncorrected ‘p’ distance for COI sequences between B. caudata of Malaysia-Thailand-China and B. caudata of Bali-Lombok was 5.65%, for 16S sequences from 2.76 to 2.99%, and for combined COI and 16S sequences 4.45 to 4.46%. The ‘p’ values are distinctly different from intraspecific ‘p’ distance (0–0.23%). Both the B. caudata lineages are distinctly separated from related species in the subgenus Zeugodacus – B. ascita, B. scutellata, B. ishigakiensis, B. diaphora, B. tau, B. cucurbitae, and B. depressa. Molecular phylogenetic analysis indicates that the B. caudata lineages are closely related to B. ascita sp. B, and form a clade with B. scutellata, B. ishigakiensis, B. diaphora and B. ascita sp. A. This study provides additional baseline for the phylogenetic relationships of Bactrocera fruit flies of the subgenus Zeugodacus. Both the COI and 16S genes could be useful markers for the molecular differentiation and phylogenetic analysis of tephritid fruit flies.

Dear Editor, Multiple infections with small liver flukes and minute intestinal flukes are the serious public health concern in the lower Mekong basin [1,2]. Although the epidemiological survey for those trematode infections are primarily carried out based on copro-parasitological examination, detection/identification of fecal eggs/worms is a tedious job and often problematic because of the morphological similarities of eggs/worms. Along with the popularization of PCR-sequencing methods, copro-DNA diagnosis and molecular phylogenetic identification/speciation have been introduced in epidemiological studies. Among various genes and non-coding lesions of nuclear and mitochondrial DNAs, mitochondrial cytochrome c oxidase subunit I (COXI) is one of the most widely used inter- and intra-species marker. Using COXI and some other markers, Lee and his colleagues performed molecular phylogenetic analyses on small liver flukes (Lee SU, Huh S. Variation of nuclear and mitochondrial DNAs in Korean and Chinese isolates of Clonorchis sinensis. Korean J Parasitol 2004; 42: 145-148) and on minute intestinal flukes (Lee SU, Huh S, Sohn WM, Chai JY. Sequence comparisons of 28S ribosomal DNA and mitochondrial cytochrome c oxidase subunit I of Metagonimus yokogawai, M. takahashii and M. miyatai. Korean J Parasitol 2004; 42: 129-135). The COX1 gene sequences appeared in those articles are; Clonorchis sinensis ({type:entrez-nucleotide,attrs:{text:AF184619,term_id:296940320,term_text:AF184619}}AF184619, {type:entrez-nucleotide,attrs:{text:AF181889,term_id:285026682,term_text:AF181889}}AF181889, {type:entrez-nucleotide,attrs:{text:AF188122,term_id:285809842,term_text:AF188122}}AF188122), Metagonimus yokogawai ({type:entrez-nucleotide,attrs:{text:AF096230,term_id:297039733,term_text:AF096230}}AF096230), Metagonimus takahashii ({type:entrez-nucleotide,attrs:{text:AF096231,term_id:297039734,term_text:AF096231}}AF096231), Metagonimus miyatai ({type:entrez-nucleotide,attrs:{text:AF096232,term_id:297039735,term_text:AF096232}}AF096232), Pygidiopsis summa ({type:entrez-nucleotide,attrs:{text:AF181884,term_id:288563287,term_text:AF181884}}AF181884), and Stellantchasmus falcatus ({type:entrez-nucleotide,attrs:{text:AF181887,term_id:285804435,term_text:AF181887}}AF181887). In addition, Park [3] compared his COXI sequence of Opisthorchis viverrini Laotian isolate (AY055 382) to those of Gymnophalloides seoi ({type:entrez-nucleotide,attrs:{text:AF096234,term_id:297039736,term_text:AF096234}}AF096234) and Neodiplostomum seoulense ({type:entrez-nucleotide,attrs:{text:AF096233,term_id:285026845,term_text:AF096233}}AF096233) registered in the DNA database (Lee et al. unpublished). For the phylogenetic analyses of COXI gene of minute intestinal flukes of our own data, we have downloaded all those above mentioned COXI of Lee et al. and aligned them including our own COXI sequence of Haplorchis taichui ({type:entrez-nucleotide,attrs:{text:EF055885,term_id:119855482,term_text:EF055885}}EF055885) [4] and Paragonimus bangkokensis ({type:entrez-nucleotide,attrs:{text:AB354227,term_id:155369203,term_text:AB354227}}AB354227) [5]. Surprisingly, those sequence data were divided into 2 distinct groups without any similarities (Fig. 1). Eventually, we realized that this astonishing result is due to the reverse complementary sequences of COXI data deposited by Lee et al. (in the bottom half of the figure). We also noticed similar mixed-up deposition of the forward and reverse sequences of COXI gene of Fasciola spp., which were also included in Fig. 1 ({type:entrez-nucleotide,attrs:{text:AJ628024,term_id:88319716,term_text:AJ628024}}AJ628024, {type:entrez-nucleotide,attrs:{text:AJ628039,term_id:88319746,term_text:AJ628039}}AJ628039, {type:entrez-nucleotide,attrs:{text:FJ469984,term_id:238631966,term_text:FJ469984}}FJ469984; Zhu XQ et al. unpublished). Fig. 1 The DNA sequence alignment of the partial COXI gene of some trematodes obtained from the GenBank. Seven sequences from the top are the forward strands with JB3 primer sequence, and 2 in the middle are the forward strands without primer. Eight sequences ... For the determination of partial COX1 sequences of Platyhelminthes, the primer set of JB3 (5'-TTT TTT GGG CAT CCT GAG GTT TAT-3') and JB4.5 (5'-TAA AGA AAG AAC ATA ATG AAA ATG-3') [6] was widely used for investigating the inter- and intra-species variations of trematodes and cestodes. We noticed the mixed-up of the forward and reverse COXI sequences by Lee et al. as well as Zhu et al. because of the presence of the characteristic feature of this primer set (boxed in Fig. 1) in the sequences. The primer sequence should be deleted from the sequence data because it is not always identical with the real DNA sequence of the gene and the inclusion of the primer sequences sometimes causes the misreading in phylogenetic analyses [7]. In 3 reverse sequences, {type:entrez-nucleotide,attrs:{text:AF181884,term_id:288563287,term_text:AF181884}}AF181884, {type:entrez-nucleotide,attrs:{text:AY055380,term_id:22203992,term_text:AY055380}}AY055380, and {type:entrez-nucleotide,attrs:{text:AF096233,term_id:285026845,term_text:AF096233}}AF096233 seems to contain also the partial sequence of the cloning vector, which should be trimmed off before deposition. In general, raw data of forward and reverse sequences obtained from the sequencer should be aligned manually by cross-checking of the wave patterns because some 10-20 bases downstream from the forward primer and upstream from the reverse primer often contain erroneous base pairs [8]. Deposition of the reverse sequence means that those sequences were not aligned against forward sequence and not quite reliable. Since each sequence data in GenBank are opened for the public use, an accuracy of the sequence data is critically important for the mutual reliability of the scientists. The scientists should aware how to deposit accurate sequence data to the DNA data base. The reappraisal and correction of those sequences mentioned above is urgently necessary.

Anopheles albertoi Unti and Anopheles arthuri Unti are revived from the synonymy with Anopheles strodei Root, and a distinct morphological form (designated in this study as Anopheles CP Form) from the Strodei Complex of Anopheles (Nyssorhynchus) is characterized. The male genitalia of An. arthuri and An. albertoi are described and illustrated for the first time. An. strodei, An. arthuri, and An. albertoi were first distinguished based on scanning electron microphotos of the eggs, and then each egg type was associated with diagnostic characters of the male genitalia. Identification of Anopheles CP Form was based on morphological characters of the male genitalia, characterized and illustrated in this study. Molecular phylogenetic analysis was most clear when an outgroup was not included, in which case using the nuclear white gene, or the white gene in combination with the mitochondrial cytochrome c oxidase subunit I (COI) gene, clearly separated these four taxa. When Anopheles quadrimaculatus Say and Anopheles stephensi Liston were included as an outgroup, combined white and COI data resolved An. strodei and An. albertoi, whereas An. arthuri was not well resolved. The single sequence of Anopheles CP Form was recovered well separated from other groups in all analyses.

Co-overexpression of the epidermal growth factor (EGF) receptor (EGFR) and c-Src frequently occurs in human tumors and is linked to enhanced tumor growth. In experimental systems this synergistic growth requires EGF-dependent association of c-Src with the EGFR and phosphorylation of Tyr-845 of the receptor by c-Src. A search for signaling mediators of Tyr(P)-845 revealed that mitochondrial cytochrome c oxidase subunit II (CoxII) binds EGFR in a Tyr(P)-845- and EGF-dependent manner. In cells this association involves translocation of EGFR to the mitochondria, but regulation of this process is ill-defined. The current study demonstrates that c-Src translocates to the mitochondria with similar kinetics as EGFR and that the catalytic activity of EGFR and c-Src as well as endocytosis and a mitochondrial localization signal are required for these events. CoxII can be phosphorylated by EGFR and c-Src, and EGF stimulation reduces Cox activity and cellular ATP, an event that is dependent in large part on EGFR localized to the mitochondria. These findings suggest EGFR plays a novel role in modulating mitochondrial function via its association with, and modification of CoxII.

The phylogeny of Paridae and allies has been studied intensively during past decades. However, the phylogenetic relationship among species tends to become increasingly controversial as different genetic markers emerge. In our study, the partial mitochondrial genes cytochrome b (cyt b) and cytochrome c oxidase subunit I (COI) were obtained from 15 species that included 10 tits, 4 long-tailed tits and a Chinese penduline tit. Analyses were conducted on the combined cyt b and COI sequences with maximum likelihood and Bayesian algorithms. Based on strong, congruent support among the different temporal partitions and models of sequence evolution, a highly resolved consensus of the relationships among Parids and their allies has been formed. The monophyly of Paridae and Remizidae is strongly supported. However, the monophyly of Paridae and Aegithalos is rejected. This agrees with previous studies using other molecular markers. Our results suggest the promotion of the subgenus Machlolophus from genus Parus to a separate genus. The phylogeny of Aegithalos is robust in the current study. However, by considering differences of both morphological and molecular characters within species, we conclude that more data are needed to define their phylogeny. Based on the patterns of taxonomic diversity and endemism, we suggest the southwestern mountain ranges of China might be the center of origin of the Aegithalos species. Divergence time estimates for the long-tailed tits range from the late Miocene to the Pleistocene (from 5.5 to 0.1 Mya) using a calibration of 2% divergence per million years. In a comparative sense, we found a congruent genetic differentiation among sympatric distribution taxa.

A specimen belonging to the deep-sea feather-star family Atelecrinidae was collected in April 2018 at the Kocebu Guyot at 1294 m deep. Based on its morphological characters, the specimen was identified as Paratelecrinuscubensis (Carpenter, 1881). This species of feather star is restricted to the deep sea and was known only from 12 records from the Bahamas and Cuba at depths of 567-892 m. The data represent the first record from the Western Pacific Ocean and the deepest record known, extending the depth where this feather star has been found to beyond 1000 m. The morphological characteristics of the Kocebu Guyot specimen were essentially identical to the morphology of the neotype, with a slight difference in the dorsal spine at the end of the cirri. The phylogenetic analysis based on the mitochondrial cytochrome c oxidase subunit I (COI), 16S rRNA genes, 28S rRNA genes, and 18S rRNA genes reveal a close relationship of P.cubensis with P.wyvilli. Results of our molecular phylogenetic analysis are consistent with our morphological identifications. Our find extends the known geographical distribution of the feather star P.cubensis to the Western Pacific Ocean and provide insights into deep-sea biodiversity in the Kocebu Guyot.

Anopheles albertoi Unti and Anopheles arthuri Unti are revived from the synonymy with Anopheles strodei Root, and a distinct morphological form (designated in this study as Anopheles CP Form) from the Strodei Complex of Anopheles (Nyssorhynchus) is characterized. The male genitalia of An. arthuri and An. albertoi are described and illustrated for the first time. An. strodei, An. arthuri, and An. albertoi were first distinguished based on scanning electron microphotos of the eggs, and then each egg type was associated with diagnostic characters of the male genitalia. Identification of Anopheles CP Form was based on morphological characters of the male genitalia, characterized and illustrated in this study. Molecular phylogenetic analysis was most clear when an outgroup was not included, in which case using the nuclear white gene, or the white gene in combination with the mitochondrial cytochrome c oxidase subunit I (COI) gene, clearly separated these four taxa. When Anopheles quadrimaculatus Say and Anopheles stephensi Liston were included as an outgroup, combined white and COI data resolved An. strodei and An. albertoi, whereas An. arthuri was not well resolved. The single sequence of Anopheles CP Form was recovered well separated from other groups in all analyses.

Three new species of the copepod family Canuellidae Lang, 1944 (Copepoda, Canuelloida) are described from the Western Pacific Ocean, based on specimens collected from coastal waters of Korea (Jeju-do Island) and the Republic of Palau. This study provides detailed morphological descriptions and illustrations for Elanella jejuensis sp. nov., Brianola coreana sp. nov., and Scottolana picrca sp. nov. Key diagnostic characters differentiating them from congeners include the unique bulbous modification of caudal ramal seta IV in E. jejuensis sp. nov., the presence of an inner seta on the P1 exopod-2 in B. coreana sp. nov., and features including reduced urosome segmentation and a modified female caudal seta II that place S. picrca sp. nov. within the longipes-group. Molecular phylogenetic analysis using mitochondrial cytochrome c oxidase subunit I (COI) and nuclear 18S ribosomal RNA (18S rRNA) sequences recovers each new species as a distinct, well-supported lineage and resolves two major clades within Canuellidae: (Elanella + Brianola) and (Scottolana + Canuella). The phylogeny further indicates that Elanella and Brianola are sister taxa, and places S. picrca sp. nov. within the longipes-group; however, the placement of S. picrca sp. nov. as sister to S. wonchoeli is weakly supported (57% bootstrap). These findings significantly advance our understanding of Canuellidae diversity and evolution in the understudied Western Pacific region.

Colour polymorphisms, and phenotypic plasticity of traits such as shells and radulae, complicate the taxonomy of many invertebrate taxa. Recent field surveys in New Zealand yielded specimens of the white cephalaspidean opisthobranch Melanochlamys lorrainae ( Rudman, 1968), not reported since the 1960s. We used an integrative approach to test whether M. lorrainae was a distinct species or an unpigmented morph of the more common black congener M. cylindrica Cheeseman, 1881. Molecular data were combined with analysis of shell morphology and observations of relative abundance and habitat preference for the two nominal species. Differences in the shape of the internal shell were congruent with body colour, but less extreme than previously reported. In field surveys, M. lorrainae was found on sand- or mudflats while M. cylindrica was primarily associated with rocky habitat and red algal turf. Molecular phylogenetic analyses were performed on three gene regions, the mitochondrial cytochrome c oxidase subunit I (COI) and large ribosomal subunit (16S) rRNA loci, and the nuclear large ribosomal subunit (28S) rRNA locus. In all analyses M. lorrainae and M. cylindrica were reciprocally monophyletic, with net genetic distances of 16.5% for COI, 2.4% for 16S and 0.9% for 28S, all comparable to distances between sister species of other cephalaspideans. The northeastern Pacific M. diomedea and an undescribed Australian species formed a clade with modest support in parsimony analysis of mtDNA, and strong support in Bayesian analysis of the nuclear 28S gene despite an unusually long branch for the Australian species. No strong phylogenetic affinity was detected between the New Zealand species and the other two Melanochlamys spp., which also differ in some morphological respects. Group mating via unilateral insemination is described for M. cylindrica.

Michajlovia turdi (Yamaguti, 1939) (Digenea: Brachylaimoidea) has been found from the Japanese thrush, Turdus cardis Temminck, 1831, in Hokkaido, the northernmost island of Japan. This is a rediscovery of M. turdi in Japan after approximately 80 years from the original description of the species as Leucochloridium turdiYamaguti, 1939. Here we redescribe the morphology of M. turdi and generate DNA barcodes for the species by sequencing nuclear ribosomal RNA (18S and 28S) and mitochondrial cytochrome c oxidase subunit I (COI) genes. Our molecular phylogenetic analysis was not effective at determining the taxonomic rank of Michajlovia in Brachylaimoidea, and consequently the genus remains as incertae sedis.

In Japan, tribe Patrobini (subfamily Patrobinae: family Carabidae) comprises five genera: Diplous, Minypatrobus, Apenetretus, Archipatrobus, and Apatrobus. We studied the phylogenetic relationships of these genera and all known species of Apatrobus using mitochondrial cytochrome c oxidase subunit I (COI) and nuclear 28S ribosomal RNA (28S) sequences. We also studied the phylogenetic placement of Japanese Patrobini taxa among those of North America and Eurasia using COI and 28S gene sequences. Among known Apatrobus taxa, Apatrobus hayachinensis and Apatrobus michinorii were phylogenetically determined not to belong to Apatrobus, but to be involved in the lineage of Apenetretus, which is known from Japan (Honshu) and Taiwan. We propose that these species be renamed Apenetretus hayachinensis n. comb. and Apenetretus michinorii n. comb., respectively. The remaining Apatrobus species are divided into eastern and western clades in Japan, with the eastern clade containing six closely related species of central Honshu and the western clade containing two groups of species found in Kyushu and in Shikoku and southwestern Honshu (Chugoku District and Kii Peninsula), respectively. Additionally, we examined the species status of some populations based on molecular phylogenetic and morphological analyses and describe the new species Apatrobus tsujii Dejima &amp; Sota, n. sp., from northern Kyushu.

Arthropod phylogeny revisited, with a focus on crustacean relationships