Freed and Cann (2013) criticized our use of linear models to assess trends in the status of Hawaiian forest birds through time (Camp et al. 2009a, 2009b, 2010) by questioning our sampling scheme, whether we met model assumptions, and whether we ignored short-term changes in the population time series. In the present paper, we address these concerns and reiterate that our results do not support the position of Freed and Cann (2013) that the forest birds in the Hakalau Forest National Wildlife Refuge (NWR) are declining, or that the federally listed endangered birds are showing signs of imminent collapse. On the contrary, our data indicate that the 21-year long-term trends for native birds in Hakalau Forest NWR are stable to increasing, especially in areas that have received active management.

We used visual observations of banded individuals and satellite telemetry from 2007 to 2011 on Hawaii Island to document movement patterns of the Hawaiian Goose (Branta sandvicensis), commonly known as Nene. Visual observations of numbered leg bands identified >19% and 10% of 323 geese at one of two breeding sites and one of two distant non-breeding areas during 2007-2011. We used satellite telemetry to document movement patterns of 10 male Nene from 2009 to 2011, and log-linear models to quantify the magnitude and individual differences in attitudinal migration. Two subpopulations of Nene moved 974.4 m (95% CI +/- 22.0) and 226.4 m (95% CI +/- 40.7) in elevation between seasons on average, from high-elevation shrublands during the non-breeding season of May August, to lower-elevation breeding and molting areas in September-April. Traditional movement patterns were thought to be lost until recently, but the movement pattern we documented with satellite telemetry was similar to attitudinal migration described by early naturalists in Hawaii prior to the severe population decline of Nene in the 20th century. Received 11 January 2011. Accepted 14 April 2012.

We compiled and analysed data from 1987?2004 on vegetation monitoring during feral ungulate management at Hakalau Forest National Wildlife Refuge, a tropical montane rainforest on the island of Hawai?i. All areas in the study had previously been used by ungulates, but cattle (Bos taurus) were removed and feral pig (Sus scrofa) populations were reduced during the study period. We monitored six line-intercept transects, three in previously high ungulate use areas and three in previously low ungulate use areas. We measured nine cover categories with the line-intercept method: native ferns; native woody plants; bryophytes; lichens; alien grasses; alien herbs; litter; exposed soil; and coarse woody debris. Vegetation surveys were repeated four times over a 16-year period. Vegetation monitoring revealed a strong increase in native fern cover and slight decreases in cover of bryophytes and exposed soil. Mean cover of native plants was generally higher in locations that were formerly lightly grazed, while alien grass and herb cover was generally higher in areas that were heavily grazed, although these effects were not statistically significant. These responses may represent early seral processes in forest regeneration following the reduction of feral ungulate populations. In contrast to many other Hawaiian forests which have become invaded by alien grasses and herbs after ungulate removal, HFNWR has not experienced this effect.

The Hakalau Forest National Wildlife Refuge was established to protect native Hawaiian forest birds, particularly endangered species. Management for forest restoration on the refuge has consisted mainly of removing feral ungulates, controlling invasive alien plants, and reforesting former pastures. To assess effects of this habitat improvement for forest birds, we estimated density annually by distance sampling and examined population trends for native and alien passerines over the 21 years since the refuge was established. We examined long-term trends and recent short-term trajectories in three study areas: (1) reforested pastureland, (2) heavily grazed open forest that was recovering, and (3) lightly grazed closed forest that was relatively intact. Three species of native birds and two species of alien birds had colonized the reforested pasture and were increasing. In the open forest, densities of all eight native species were either stable or increasing. Long-term trends for alien birds were also generally stable or increasing. Worryingly, however, during the most recent 9 years, in the open forest trajectories of native species were decreasing or inconclusive, but in the reforested pasture they generally increased. The closed forest was surveyed in only the most recent 9 years, and trajectories of native species there were mixed. Overall, long-term population trends in Hakalau are stable or increasing, contrasting with declines in most other areas of Hawai'i over the same period. However, more recent mixed results may indicate emergent problems for this important bird area.

1 Hawai‘i Cooperative Studies Unit, University of Hawai‘i at Hilo, Pacific Aquaculture and Coastal Resources Center, P.O. Box 44, Hawai‘i National Park, HI 96718, USA 2 U.S. Geological Survey, Pacific Island Ecosystems Research Center, P.O. Box 44, Hawai‘i National Park, HI 96718, USA 3 U.S. Fish and Wildlife Service, Hakalau Forest National Wildlife Refuge, 60 Nowelo St., Suite 100, Hilo, HI 96720, USA 4 Current address: Department of Environmental Studies, University of New England, 11 Hills Beach Road, Biddeford, ME 04005, USA

We compiled and analyzed data from 1987 to 2004 on feral pig (Sus scrofa) population indices affected by control methods at Hakalau Forest National Wildlife Refuge, a tropical montane rainforest on the island of Hawai`i. These population data included annual sign surveys, the number of pigs removed from fenced management units, and age and reproductive status from necropsies. There was an even sex ratio (1 female:1.02 males) within the population and within age classes. Boars lived to 60 months while sows lived to 48 months. Pregnancy occurred throughout the annual cycle, but lactation peaked in April– June. Reproductive rates also increased with age, peaking at 2–4 years in sows. We reconstructed the standing population within a 2,024-ha closed unit to examine demographic processes. We estimated that annual removal of >41–43% of the population was necessary to affect a decline. Annual sign surveys showed a strong and sustained decline in pig activity after 1997 relative to unmanaged areas. When compared with staff or public hunting, snaring was the most efficient control method. TRANSACTIONS OF THE WESTERN SECTION OF THE WILDLIFE SOCIETY 42:53–67; 2006

AbstractThe Akiapolaau (Hemignathus munroi), an endangered Hawaiian honeycreeper, drills 3–5 mm deep holes in particular ohia trees (Metrosideros polymorpha) and drinks the sap that emerges, a remarkable example of convergent evolution in sap-feeding behavior with mainland woodpeckers and Australian sugar-gliders. There has been little research on how this species selects sap trees (“Aki trees”) and what advantages they confer. We marked the locations of and collected sap samples and microhabitat data from 101 Aki trees and 73 randomly selected control trees in Hakalau Forest National Wildlife Refuge, Hawaii. Aki trees were rare (2 ha−1), spatially clustered, and defended by Akiapolaau. Sap flow volume and speed were substantially greater in Aki trees than in controls. Aki trees also were larger, had thinner bark, and were more likely to be located on convex east-facing slopes where more light is available. Those results support the hypothesis that Aki trees are selected on the basis of high sap flow and a suite of unique microhabitat and tree characteristics. Sap may be an important energy source in times of low insect availability and a potent alternative to nectar for the Akiapolaau. Aki trees are both a fascinating example of niche specialization and a factor that should be considered when conserving or restoring habitat for this endangered species.

The Hawaiian endemic mints, which comprise 58 species of dry-fruited Haplostachys and fleshy-fruited Phyllostegia and Stenogyne, represent a major island radiation that likely originated from polyploid hybrid ancestors in the temperate North American Stachys lineage. In contrast with considerable morphological and ecological diversity among taxa, sequence variation in the nrDNA 5S non-transcribed spacer was found to be remarkably low, which when analyzed using standard parsimony resulted in a lack of phylogenetic resolution among accessions of insect-pollinated Phyllostegia and bird-pollinated Stenogyne. However, many within-individual nucleotide polymorphisms were observed, and under the assumption that they could contain phylogenetic information, these ambiguities were recoded as new character states. Substantially more phylogenetic structure was obtained with these data, including the resolution of most Stenogyne species into a monophyletic group with an apparent recent origin on O'ahu (3.0 My) or the Maui Nui island complex (2.2 My). Subsequent diversification appears to have involved multiple inter-island dispersal events. Intergeneric placements for a few morphotypes, seemingly misplaced within either Phyllostegia or Stenogyne, may indicate reticulation as one polymorphism-generating force. For a finer scale exploration of hybridization, preliminary AFLP fragment data were examined among putative hybrids of Stenogyne microphylla and S. rugosa from Mauna Kea, Hawai'i, that had been identified based on morphology. Cladistic analysis (corroborated by multivariate correspondence analysis) showed the morphologically intermediate individuals to group in a strongly supported monophyletic clade with S. microphylla. Therefore, reticulation could be both historic and active in Stenogyne, and perhaps a force of general importance in the evolution of the Hawaiian mints. The relatively greater extent of lineage-sorted polymorphisms in Stenogyne may indicate selective differentiation from other fleshy-fruited taxa, perhaps through the agency of highly specialized bird pollinators that restricted gene flow with other Hawaiian mint morphotypes.

Degradation of type I collagen, the most abundant collagen, is initiated by collagenase cleavage at a highly conserved site between Gly775 and I1e776 of the od(I) chain. Mutations at or around this site render type I collagen resistant to collagenase digestion in vitro. We show here that mice carrying a collagenaseresistant mutant Colla-1 transgene die late in embryogenesis, ascribable to overexpression of the transgene, since the same mutation introduced into the endogenous Colla-1 gene by gene targeting permitted normal development of mutant mice to young adulthood. With increasing age, animals carrying the targeted mutation developed marked fibrosis of the dermis similar to that in human scleroderma. Postpartum involution of the uterus in the mutant mice was also impaired, with persistence of collagenous nodules in the uterine wall. AIthough type I collagen from the homozygous mutant mice was resistant to cleavage by human or rat fibroblast collagenases at the helical site, only the rat collagenase cleaved collagen trimers at an additional, novel site in the nonhelical N-telopeptide domain. Our results suggest that cleavage by murine collagenase at the N-telopeptide site could account for resorption of type I collagen during embryonic and early adult life. During intense collagen resorption, however, such as in the immediate postpartum uterus and in the dermis later in life, cleavage at the helical site is essential for normal collagen turnover. Thus, type I collagen is degraded by at least two differentially controlled mechanisms involving collagenases with distinct, but overlapping, substrate specificities. T YPE I collagen is among the most abundant components of the extracellular matrix of many tissues, particularly in skin, tendons, ligaments, uterus, large blood vessels and bone. The helical trimeric molecules of type I collagen comprise two al(I) chains and one ~2(I) chain, encoded by two separate genes, Colla-1 and Colla-2, respectively (50). In mice, type I collagen is first synthesized in the mesenchymal stroma of the head, heart and somites at day 8 of gestation (E8) and its production continues throughout development and postnatal life (27). It has been demonstrated that type I collagen is critical for bone development (9, 10), hematopoiesis (29, 37), integrity of the vascular system (29) and for mesenchymal-epithelial induction in organogenesis (6). The collagen content of different tissues during development and in adult animals is tightly regulated by coordinated processes of synthesis and degradation (2, 8, 57). Failure to maintain an equilibrium between synthesis and degradation leads to diverse human connective tissue disorders characterized by excessive resorption (e.g. os-

The membranes surrounding the chick embryo undergo striking morphological changes before hatching, which include structural degradation of the allantoic membrane. The fibrillar collagen content of the membranes declined by embryonic day (ED) 20 (the day of hatching). By ED 19, a 55-kDa matrix metalloproteinase (MMP) activity appeared in the extraembryonic fluid, and by ED 20 there was substantial 55-kDa MMP activity in embryonic membrane extracts. Reverse transcription-polymerase chain reaction was employed to clone a partial cDNA representing the chicken homologue of MMP-13, a 55- to 57-kDa enzyme. MMP-13 mRNA dramatically increased in abundance in embryonic membranes by ED 19, reaching a peak on ED 20. Introduction of the MMP inhibitor batimastat into the extraembryonic fluid prevented the structural changes in the embryonic membranes before hatching. We conclude that, like mammalian fetal membranes, chick embryonic membranes undergo terminal remodeling before hatching, in part as a result of increased MMP activity. The chicken egg system represents a novel in vivo model for exploring biochemical events leading to embryonic membrane remodeling prior to birth and to test inhibitors of MMPs for their ability to prevent collagenolysis and fetal membrane rupture.

The regulation, by progesterone, of serotonin-induced interleukin-1alpha production was studied in primary cultures of rat uterine smooth muscle cells. Prior reports from this laboratory have demonstrated that these cells produce IL-1alpha and IL-1beta mRNAs in response to the hormonal action of serotonin. Results of the present study indicate that treatment of myometrial smooth muscle cells with medroxyprogesterone acetate (MPA) results in a marked decrease in IL-1alpha protein as measured by western blot analysis. These decreases occur even in the presence of maximally-inducing concentrations of serotonin. MPA-mediated changes in IL-1alpha protein are characterized by a rapid decline in IL-1alpha mRNA levels. This inhibition by medroxyprogesterone also occurs when cells are stimulated to produce IL-1alpha by PMA rather than serotonin. Thus, when cells are cultured in the presence of both inducer and inhibitor, the inhibitor, progesterone, clearly dominates in the control of IL-1alpha expression. This effect is concentration-dependent, can be mimicked by native progesterone or glucocorticoids, but is unaffected by estradiol. The ability of progestins to decrease IL-1alpha mRNA is blocked by both inhibitors of transcription and translation and by treatment with RU-486. Progesterone had no effect on chloramphenicol acetyl transferase (CAT) transcription from two different IL-1alpha promoter constructs, indicating that progesterone's action appears to be dependent on post-transcriptional rather than transcriptional regulation. Conversely, progesterone accelerated the normal rate of decay of IL-1alpha mRNA that occurs following the removal of serotonin from the cultures. These results suggest that progesterone decreases IL-1alpha levels by stimulating the production of an intracellular intermediate that decreases the stability of IL-1alpha mRNA.

Serotonin (5-hydroxytryptamine (5-MT)) has been shown to be a regulator of gene expression in rat myometrial smooth muscle cells (SMC). Serotonin activates the genes for interstitial collagenase, interleukin-1α, -1β and interleukin-6, among others. On the other hand, serotonin down-regulates the genes for types I and III collagen and fibronectin. Here we show that serotonin is also a negative regulator of the expression of anti-protease α2-macroglobulin (α2M) in SMC. The serotonin-dependent repression occurs at both the mRNA and protein levels, and is mediated by the 5-HT2A receptor subtype. The inhibitory effect is prevented by cycloheximide, indicating the requirement for the synthesis of one or more proteins. Interleukin-1 (IL-1), which is induced by serotonin in SMC and is required for subsequent interstitial collagenase induction, appears not to be one of these intermediates. On the other hand, progesterone, the major steroid hormone of pregnancy, is capable of reversing the serotonin-mediated inhibition of α2M. The phorbol myristate acetate (PMA), which mimics the induction of interstitial collagenase by serotonin, fails to affect the inhibition of α2M production. The cell-permeable cyclic AMP analogue 8-bromoadenosine 3′:5′-cyclic monophosphate sodium salt (8-bromo-cAMP), is, however, capable of fully reproducing the action of serotonin on α2M. These results further speak to the ability of serotonin to regulate gene expression in the myometrial SMC, both positively and negatively. In addition, although all the effects of serotonin so far identified are mediated by the 5-HT2A receptor, different post-receptor pathways appear to mediate the positively and negatively regulated genes.

The activation of the gene for interstitial collagenase in myometrial smooth muscle cells is absolutely dependent upon the presence of serotonin. Our previous studies investigating the mechanisms of this induction demonstrated that the mRNAs of both interleukin-1 (IL-1) isoforms, IL-1 alpha and IL-1 beta, are induced by serotonin and that the induction of IL-1 is required for the subsequent induction of collagenase. These data provided compelling evidence that serotonin-induced IL-1 acts via an autocrine loop in activating the collagenase gene. The experiments described here were designed to examine the potential role of each IL-1 isoform in collagenase production by using neutralizing antisera specific to each isoform of the cytokine, The antisera were examined for their ability to inhibit the serotonin-dependent production of the mRNA for collagenase and of the cytokines themselves. Neutralizing antiserum against IL-1 alpha, but not against IL-1 beta, inhibited the induction of the mRNA for collagenase and of the mRNAs for both IL-1 alpha and IL-1 beta, Western analysis indicated that detectable levels of IL-1 alpha protein, but not that of IL-1 beta, are produced at the time of serotonin-dependent collagenase induction. In contrast, significant levels of IL-1 beta protein are detected only when bacterial lipopolysaccharide is added to the cells. Taken together, the results of our study indicate that IL-1 alpha, but not IL-1 beta, plays an obligatory role in multiple serotonin-mediated gene regulations in the myometrial smooth muscle cell. In addition, the data suggest that IL-1 beta production has the potential for modifying myometrial function in pathological settings, particularly that of uterine infection.

Serotonin (5-hydroxytryptamine (5-MT)) has been shown to be a regulator of gene expression in rat myometrial smooth muscle cells (SMC). Serotonin activates the genes for interstitial collagenase, interleukin-1α, -1β and interleukin-6, among others. On the other hand, serotonin down-regulates the genes for types I and III collagen and fibronectin. Here we show that serotonin is also a negative regulator of the expression of anti-protease α2-macroglobulin (α2M) in SMC. The serotonin-dependent repression occurs at both the mRNA and protein levels, and is mediated by the 5-HT2A receptor subtype. The inhibitory effect is prevented by cycloheximide, indicating the requirement for the synthesis of one or more proteins. Interleukin-1 (IL-1), which is induced by serotonin in SMC and is required for subsequent interstitial collagenase induction, appears not to be one of these intermediates. On the other hand, progesterone, the major steroid hormone of pregnancy, is capable of reversing the serotonin-mediated inhibition of α2M. The phorbol myristate acetate (PMA), which mimics the induction of interstitial collagenase by serotonin, fails to affect the inhibition of α2M production. The cell-permeable cyclic AMP analogue 8-bromoadenosine 3′:5′-cyclic monophosphate sodium salt (8-bromo-cAMP), is, however, capable of fully reproducing the action of serotonin on α2M. These results further speak to the ability of serotonin to regulate gene expression in the myometrial SMC, both positively and negatively. In addition, although all the effects of serotonin so far identified are mediated by the 5-HT2A receptor, different post-receptor pathways appear to mediate the positively and negatively regulated genes.

Differential Regulation of matrix metalloproteinase gene expression by serotonin and progesterone in rat uterine smooth muscle cells. † 294

The cell-surface localization and site of activation of type IV collagenases/gelatinases (matrix metalloproteinases, MMP) in bovine pulmonary microvascular endothelial (BPMVE) cells was examined. Sucrose density centrifugation of plasma membranes and immunofluorescent staining of whole cells indicated association of 72 kDa (MMP-2) and 96 kDa (MMP-9) type IV collagenase/gelatinases with the plasma membrane. Incubation of the BPMVE cells with rhodaminated MMP-9 demonstrated colocalization with beta 1-integrin, indicating incorporation into the focal contacts. The focal contacts were extracted with saponin, and associated proteolytic activity was examined by zymography. The focal contacts contained latent MMP-2, and stimulation of the cells with cytochalasin D or with 8-bromoadenosine 3',5'-cyclic monophosphate with 3-isobutyl-1-methylxanthine increased both latent and activated MMP-9 in the focal contacts. Addition of these stimuli in unconditioned culture medium did not produce this effect, indicating that the MMP-9 in focal contact extracts was derived from previously secreted enzyme. The activated metalloproteinase degraded extracellular matrix collagens and was inhibited by 1,10-phenanthroline. These findings indicate that endothelial cells release MMP into the extracellular milieu and then concentrate and activate MMP-9 from medium at the focal contacts.

Platelet-derived growth factor (PDGF), a bone cell mitogen, stimulates bone collagen degradation and does not enhance bone matrix apposition rates. The mechanism of the effect on collagen degradation is unknown, and it could involve changes in interstitial collagenase synthesis. We tested the effects of PDGF on interstitial collagenase expression in cultures of osteoblast-enriched cells from fetal rat calvariae (Ob cells). After 4-8 h of treatment, PDGF BB at 0.3 nM increased steady state collagenase messenger RNA (mRNA), whereas PDGF AA had no effect. The effect of PDGF BB on collagenase transcripts was dose dependent. PDGF BB increased the levels of immunoreactive collagenase after 6 h, whereas the levels were decreased after 16 h. Stimulation of collagenase mRNA by PDGF BB was dependent on de novo protein synthesis and activation of protein kinase C. PDGF BB prolonged the half-life of collagenase mRNA in transcriptionally arrested cells. PDGF BB initially increased and subsequently decreased the rate of collagenase gene transcription and the levels of collagenase heterogeneous nuclear RNA. In conclusion, PDGF BB regulates interstitial collagenase in Ob cells by transcriptional and posttranscriptional mechanisms, and this effect may contribute to its stimulatory actions on bone collagen degradation.

Basic fibroblast growth factor (bFGF) is a bone cell mitogen that affects osteoblastic function by suppressing type I collagen synthesis. The investigators in this study examined whether bFGF also regulates interstitial collagenase and tissue inhibitors of metalloproteinases (TIMPs) in osteoblast-enriched cells isolated from 22-day fetal rat calvariae. After exposure to 600 pM bFGF, interstitial collagenase messenger RNA (mRNA) levels, as determined by Northern hybridization analysis, increased after 2 h and were maximally stimulated to approximately 13-fold at 6 h. Exposure of osteoblast-enriched cells to 0.06-6 nM bFGF increased collagenase mRNA in a dose-dependent manner, and bFGF also increased immunoreactive collagenase measured in the culture medium by Western blot analysis. The protein synthesis inhibitor cycloheximide, as well as two inhibitors of protein kinase C, staurosporine and sangivamycin, prevented the bFGF induction of collagenase transcripts, whereas indomethacin, an inhibitor of prostaglandin synthesis, decreased the effect of bFGF on collagenase mRNA levels by about 50%. After exposure to 600 pM bFGF, levels of TIMP 1 and TIMP 3 mRNAs were also maximally stimulated to approximately 6-fold at 16 h and 4-fold at 6 h. bFGF did not modify TIMP 2 expression. In conclusion, bFGF may modulate degradation of collagenous bone matrix by inhibiting collagen as well as stimulating collagenase and TIMPs by osteoblasts.

We found that the matrix metalloproteinases collagenase (MMP-1) and stromelysin (MMP-3) each has the ability to degrade a novel substrate, serum amyloid A (SAA3). SAA3 is a product of rabbit synovial fibroblasts stimulated with phorbol esters or interleukin-1, and it acts in an autocrine or paracrine manner to induce collagenase in both rabbit and human fibroblasts. Recombinant rabbit fibroblast procollagenase and human fibroblast prostromelysin were produced by baby hamster kidney (BHK) cells stably transfected with these genes, and latent enzyme was activated with aminophenylmercuric acetate (APMA). The Km for both enzymes was ∼ 10 μM, and the Vmax for collagenase was ∼ 6 pmol/minute/100 ng enzyme, while that for stromelysin was about 3-fold faster. Treatment of SAA3 with either enzyme generated a fragment of approx. 6 kDa that has the same amino terminus as the parent molecule, but this fragment was rapidly degraded. We have been unable to isolate C-terminal fragments, suggesting that the mature protein is cleaved at multiple sites and/or that the initial cleavage fragment is readily digested. The amino acid composition of the 6 kDa fragment suggests that the 14kDa protein is cleaved at residues 50–57, a hydrophobic region that is conserved between rabbit SAA3 and human SAA1. We conclude that the ability of collagenase and stromelysin to degrade SAA3 broadens the repertoire of substrates for these matrix degrading enzymes, and we speculate that the presence of a feedback mechanism that can subvert the autocrine/paracrine stimulation of matrix-degrading enzymes may play a role in limiting degradation during inflammatory conditions.