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Recent concerns of declining bee populations have highlighted the importance of monitoring wild bees, but bee community assessments are hampered by species complexes that are difficult to identify. Bees in the genus Melissodes are often considered challenging to identify to species, with two widespread North American species, M. agilis Cresson and M. trinodis Robertson, being particularly difficult due to similar morphology, geographic ranges, and preferred floral hosts. These two species exhibit characteristics of cryptic species complexes, raising the possibility that our current understanding of their taxonomy is incomplete. We conducted a study to clarify the species boundaries within this complex, and to test if geometric morphometrics could be used to differentiate its member taxa. We sequenced fragments of the mitochondrial COI gene from 112 M. agilis/trinodis specimens, and integrated them into a phylogeny based on published reference sequences of over 70 Melissodes species. We additionally landmarked forewing venation for 102 of these specimens, tested if forewing morphometrics was associated with sex and phylogenetic clade, and tested if forewing morphometrics could accurately assign specimens to their proper clade and sex. Phylogenetic reconstructions resulted in nearly all specimens being assigned to three primary clades, with one clade containing reference sequences for M. agilis and M. trinodis, and two clades appearing to be undetermined cryptic taxa. Forewing morphometrics differed between clades and sexes, and was able to assign specimens to their proper clade or sex with over 80% accuracy, although accuracy of classification to clade declined to between 33-93% after cross-validation. Our results suggest the existence of cryptic diversity within M. agilis and M. trinodis, and indicate that forewing morphometrics can characterize some of this diversity. M. agilis and M. trinodis may comprise a complex of 3-5 cryptic species, but whether these are described or undescribed species is unknown. Also unclear is the degree to which the potential cryptic species contribute to the economically important sunflower pollination services currently considered to be conducted by M. agilis and M. trinodis. We encourage additional study of this complex to determine the nature of this cryptic diversity and resolve the taxonomic questions this study has raised.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23944号 / 農博第2493号 / 新制||農||1090(附属図書館) / 学位論文||R4||N5379(農学部図書室) / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 德地 直子, 教授 北山 兼弘, 教授 神﨑 護, 准教授 伊勢 武史 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The G protein-coupled estrogen receptor GPER/GPER1, also known as GPR30, was originally cloned as an orphan receptor and later shown to be specifically activated by 17-ß-estradiol. This has led to its classification as an estrogen receptor and expanded the perspective on the mechanisms underlying the rapid estrogenic effects reported over the years. GPER is strongly expressed in the central nervous system and peripheral tissues and appears to be involved in a wide variety of physiological and pathological processes. Estrogens are known to alter the processing of nociceptive sensory information and analgesic responses in the central nervous system. Both analgesic and pro-nociceptive effects of estrogens have been reported. Some pro-algesic estrogenic responses have a short latency, suggesting a non-genomic mechanism of action. Immunohistochemical studies in rodents prove the existence of GPER in pain-relevant areas of the nervous system such as dorsal root ganglia, superficial dorsal horn of the spinal cord, periaqueductal gray (PAG), amygdala, trigeminal sensory nucleus and thalamus. In the periphery, activation of GPER results in pro-nociceptive effects. However, GPER involvement in pain processing at central levels is largely unexplored. Thus, the work presented in this thesis was aimed at investigating whether GPER modulates nociception at spinal and supraspinal sites. The behavioral response to GPER activation in the spinal cord and PAG was evaluated in an acute grooming test (scratching, biting and licking behavior) and in the hot plate test, respectively. Intrathecal challenge of mice with the GPER agonist G-1 (0.1-1 nmol) induced a dose-dependent increase in pain-related behaviors, that was abolished by pre-treatment with the GPER antagonist G15 (1-10 nmol), confirming GPER specificity of the response. Likewise, intra-PAG microinjection of G-1 (10-100 pmol) to rats reduced the nociceptive threshold in the hot plate test, an effect that was G15 sensitive. To obtain further insight on the mechanisms involved in the behavioral effects observed in whole animals, we tested the effect of GPER ligands on neuronal membrane potential, intracellular calcium concentration ([Ca2+]i) and reactive oxygen species (ROS) accumulation. The membrane depolarization and the increases in [Ca2+]i and ROS levels are markers of neuronal activation, underlying pain sensitization in the spinal cord and pain facilitation in the PAG. Electrophysiological recordings from superficial dorsal horn and lateral PAG neurons indicate neuronal depolarization upon G-1 application, an effect that was fully prevented by G15 pre-treatment. Both cultured spinal neurons and cultured PAG neurons responded to G-1 administration by elevating [Ca2+]i and mitochondrial and cytosolic ROS levels. In the presence of G15, G-1 did not elicit the calcium and ROS responses. Collectively, these results demonstrate that GPER modulates both the ascending and descending pain pathways to increase nociception via cytosolic calcium elevation and ROS accumulation in spinal and PAG neurons, respectively. These findings broaden the current knowledge on GPER involvement in physiology and pathophysiology, providing the first evidence of its pro-nociceptive effects at central levels and characterizing some of the mechanisms involved. Moreover, we show for the first time ROS accumulation downstream of GPER activation, extending the current understanding of GPER signaling. / Pharmacology

<p> Caenorhabditis elegans (C. elegans) is a model organism which is known for its transparent body, small body size, high reproductivity and short lifecycle. Several important genes and signal transduction pathways are well conserved in C. elegans. lin//, a LIM homeobox family member, plays a crucial role in the development of the vulva in C. elegans. LIM homeobox genes are a subgroup of Homeobox family that play fundemental role in animal development. In C. elegans lin-If mutant animals fail to form a functional vulva and vulval-uterine connection and consequently exhibit egg-laying defective phenotype. The cell lineage and marker gene expression studies have shown that lin-// is required for the patterning of all primary and secondary lineage vulval cells. lin- II also functions in the nervous system. </p> <p> lin-// expression is mainly observed in the developing vulval cells and in the pi cells which are involved in the formation of vulval-uterine connection. lin-If expression is also seen in VCs and in some of the head and tail neurons. The completed genome sequences of closely related species in Caenorhabditis genus serve as a power tool to do systematic comparative studies. The lin-If regulatory sequences from these species have been compared along with the expression patterns. </p> <p> We looked at the regulation of lin-// in closely related nematode species like C. elegans, C. briggsae, C. remanei and Caenorhabditis n species. </p> <p> Consistent with this. expression of lin-11 is observed in the developing vulval cells. We are interested in understanding evolutionary changes in the regulation and function of lin-II in reproductive system </p> <p> /in-11 is a LIM homeodomain family member which IS involved in several developmental events. lin-11 role is documented in the thermoregulatory circuit specifying AIY interneuron, in chemosensory neurons like A W A and olfactory neurons A WS. During vulval development lin-II expression is dynamically expressed in subset of secondary lineage cells and is broadly expressed in all the cells indicating its role in cell identity and cell fusion of the vulval cells. lin-II is also required for the formation of vulval uterine connection which is the passage to lay eggs in the hermaphrodite. linllloss of function hermaphrodites have change in the axis of the secondary lineage cells during vulval development, uterine Jt cell migration defect, defects in the AIY, A W A and A WS interneurons resulting in egg-laying defect and protruding vulva and neuronal defects and reduced mating efficiency. </p> <p> The expression pattern of lin-If in closely related species is highly similar but not identical. From the sequence comparison of lin-If regulatory sequences a 1 kb conserved block of sequences have been identified which includes the regulatory sequences responsible for the expression of lin-If in vulva and Jt cells. We propose that cisregulatory elements controlling lin-If gene expression are slowly evolving though there is no change in the function which indicates that lin-If plays critical role during the development of the vulva and other tissues. </p> / Thesis / Master of Science (MSc)