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Review of Canadian species of the genus Dinaraea Thomson, with descriptions of six new species (Coleoptera, Staphylinidae, Aleocharinae, Athetini)

Abstract Twelve species of the genus Dinaraea Thomson are recognized in the Nearctic region, ten of which occur in Canada, all east of the Rocky Mountains. Six species are herein described as new to science: Dinaraea bicornis Klimaszewski & Webster, sp. n.; Dinaraea curtipenis Klimaszewski & Webster, sp. n.; Dinaraea longipenis Klimaszewski & Webster, sp. n.; Dinaraea quadricornis Klimaszewski & Webster, sp. n.; Dinaraea worki Klimaszewski & Jacobs, sp. n.; and Dinaraea piceana Klimaszewski & Jacobs, sp. n. Four formerly described species are confirmed in Canada: Dinaraea angustula (Thomson), Dinaraea backusensis Klimaszewski & Brunke, Dinaraea borealis Lohse, and Dinaraea pacei Klimaszewki & Langor. The previously unknown male of Dinaraea borealis Lohse and female of Dinaraea backusensis are described. All species are illustrated with colour habitus images and black and white images of the median lobe of the aedeagus and spermatheca, and tergite VIII and sternite VIII of both sexes. New habitat and distribution data are presented and a key to all Nearctic species of the genus is provided.

rected posteriad in apical two-thirds, anteriad in basal third, and laterad at sides; elytra short, at suture as long as pronotum or only slightly longer; abdominal tergites 3-5 each with distinct basal impression; tarsal formula 4-5-5; male tergite VIII in majority of species with small teeth at apical margin and sometimes also in adjacent area of disc; median lobe of aedeagus with athetine bridge and of a simple form, with inconspicuous structures of internal sac; spermatheca with conical capsule and long slim stem coiled posteriorly.
Comments. Based on the morphology of the male tergite VIII, two groups of species are recognized. One group, consisting of the majority of species, is characterized by tergite VIII bearing two-to-several small teeth on the apical margin. Species in the second group (D. borealis, D. curtipenis, D. longipenis, D. planaris, D. subdepressa), have tergite VIII truncate and without teeth on the apical margin.
Biology. Many species are known from the subcortical galleries of other insects but their diet is unknown.

1
Elytra at suture shorter than pronotum (Figs 1a, 4a, 10a Pronotum trapezoidal in shape, broadest in apical third, punctures very dense, pronotum appearing matte (Fig. 1a); male tergite VIII with two teeth (Fig. 1c); median lobe of aedeagus and spermatheca as illustrated (Fig. 1b Pronotum rectangular in shape, broadest at middle, punctures very dense (pronotum appearing matte) and distanced from each other by a diameter of a puncture (Fig. 2a); male tergite VIII, median lobe of aedeagus, and spermatheca as illustrated ( Fig. 2c,  Body dark brown with reddish tinge (Fig. 8a); tubus of median lobe of aedeagus long and straight ventrally, and with narrow sclerites of internal sac (Fig. 8b); spermatheca as illustrated (Fig. 8e)  Pronotum dark brown and elytra yellowish-brown (Fig. 5a); male tergite VIII, median lobe of aedeagus and spermatheca as illustrated (Fig. 5b,  Median lobe of aedeagus with tubus slightly sinuate apically and slightly produced ventrally at apex, internal sac structures short and straight (Fig. 13a); male tergite VIII without apical teeth (Fig. 13b), sternite VIII as illustrated (Fig. 13c) Lohse and Smetana (1985), there are no major external differences between the two species and they key to the same couplet. Etymology. The specific name bicornis means 'with two horns' in allusion to the two teeth on the male tergite VIII.
Description. Body length 3.1-3.4 mm; body dark brown with at least posterior part of elytra paler, with legs, antennae and labial palpi yellowish-brown; head and pronotum with dense microsculpture and appearing matte; elytral and particularly pronotal microsculpture less dense and their integument appears glossy; head about as broad as pronotum, genae slightly longer than eyes in dorsal view; pronotum broadest in apical third, slightly transverse, longer than elytra at suture; elytra transverse, shorter than pronotum at suture, truncate posteriorly; abdomen arcuate laterally, broadest in apical third; male tergite VIII with two small sharp medial teeth at apical margin ( Fig.  1c), sternite VIII slightly produced posteriorly, antecostal suture arcuate (Fig. 1d); median lobe of aedeagus with short and straight venter of tubus and narrowly rounded apex (Fig. 1b); female tergite VIII slightly sinuate apically on each side of disc ( Fig. 1f), sternite VIII rounded apically, antecostal suture sinuate (Fig. 1g); spermatheca with pear-shaped capsule and moderately deep apical invagination, stem narrow, long and looped posteriorly, slightly swollen at apex (Fig. 1e).
Distribution. Known from New Brunswick and Ontario. Collection and habitat data. Most adults were collected in April and May, and one specimen in October. The New Brunswick specimens were found in moss and leaf litter near the edges of small vernal pools in forests containing eastern white cedar (Thuja occidentalis L.) and red maple (Acer rubrum L.), and sometimes alder and poplar.  Etymology. The specific name quadricornis, means 'with four horns' in allusion to the four teeth on the male tergite VIII.

Dinaraea quadricornis
Diagnosis. Dinaraea quadricornis (habitus Fig. 2a) may be distinguished from congeners by the following combination of characters: body length 3.2-3.5 mm; head, pronotum and elytra matte with dense microsculpture; pronotum broadest at middle; elytra at suture as long as pronotum, with dense punctation similar to that on pronotum; antennal articles 7-10 strongly transverse; male tergite VIII with four small sharp apical teeth (Fig. 2c); median lobe of aedeagus with straight and short tubus, narrowly rounded apically and slightly produced ventrally (Fig. 2b); spermatheca with pear-shaped capsule and moderately deep apical invagination, stem narrow, long and looped posteriorly, slightly swollen at apex (Fig. 2e).
Distribution. Known only from New Brunswick. Collection and habitat data. Adults were collected from March to September in several microhabitats: under tight bark of white elm in a silver maple (Acer saccharinum L.) forest; in a gilled mushroom located on a ridge with oaks in a silver maple forest; in a hardwood forest with silver maple and butternut (Juglans cinerea L.); under the bark of a hardwood tree; in a wood pile; under the bark of spruce (Picea sp.) in a mixed forest; under the bark of a spruce log in an old mixed forest; in a hardwood forest under the bark of a beech (Fagus grandifolia Ehrh.) log; in decaying mushrooms in a mixed forest with red spruce (Picea rubens Sarg.) and yellow birch (Betula alleghaniensis Britt.); and in a red oak (Quercus rubra L.) forest under the bark of red oak. Flying adults were also captured in Lindgren funnel traps in an old red oak forest and in an old silver maple forest and seasonally flooded marsh. One individual was sifted from drift material (tree buds) in an eddy area along a fast-flowing, clear, cold and rocky river. Diagnosis. Dinaraea backusensis (habitus Fig. 3a) may be distinguished from congeners by the following combination of characters: body length 2.4-2.6 mm; head, pronotum and elytra moderately glossy with dense microsculpture; pronotum broadest in about apical third and narrowest at base; elytra at suture as long as pronotum, with dense punctation similar to that on pronotum; antennal articles 7-10 moderately transverse; male tergite VIII with four small apical median teeth, median ones rounded (Fig. 3d); median lobe of aedeagus with straight and short tubus, narrowly rounded apically (Fig. 3b, h); spermatheca with large pear-shaped capsule and moderately shallow apical invagination, stem moderately long and looped posteriorly, apical end strongly swollen (Fig. 3c).
Distribution. Known from Ontario and New Brunswick in Canada and Massachusetts in the USA. Collection and habitat data. Adults were collected from March to August mostly from under the bark of rotting logs (spruce, maples) in a variety of hardwood and mixed hardwood-conifer forest types. Some adults were found in polypore fungi on a large fallen basswood (Tilia americana L.) and in moss and litter at the base of cedar in an old-growth eastern white cedar swamp. Flying adults were also captured in Lindgren funnel traps in a red spruce forest with red maple and balsam fir (Abies balsamea (L.) Mill.) and in an old red pine (Pinus resinosa Ait.) forest.
Material  Etymology. Named for Dr. Timothy Work, Université du Québec à Montréal, Quebec, Canada, who provided many insect specimens for this study and who has advanced the ecological knowledge of epigaeic beetles in Canada.

Dinaraea worki
Diagnosis. Dinaraea worki (habitus Fig. 4a) may be distinguished from congeners by the following combination of characters: body length 2.3-2.6 mm; head, pronotum and elytra moderately glossy with moderately dense microsculpture; pronotum broadest in middle and narrowest at base; elytra at suture as long as pronotum, with dense punctation similar to that on pronotum; antennal articles 7-10 strongly transverse; male tergite VIII with four small apical teeth, all short and rounded (Fig. 4c); median lobe of aedeagus with straight and short tubus narrowly rounded apically (Fig. 4b); spermatheca with large pear-shaped capsule and long apical invagination, stem short and looped posteriorly, with slightly swollen apical part (Fig. 4e).
Description. Body length 2.3-2.6 mm; body dark brown with legs, antennae (at least basally), labial palpi and posterior part of elytra yellowish-or reddish-brown; head, pronotum and elytra moderately glossy, with moderately dense microsculpture; abdominal microsculpture moderately dense and integument more glossy; head about as broad as pronotum, genae slightly longer than eyes in dorsal view; pronotum broadest in middle, slightly transverse, about as long as elytra at suture; elytra transverse, truncate posteriorly; abdomen arcuate laterally, broadest in middle; male tergite VIII with four small rounded teeth at apical margin (Fig. 4c), sternite VIII rounded posteriorly, antecostal suture arcuate and anterior margin broadly arcuate (Fig. 4d); median lobe of aedeagus with short and straight venter of tubus and narrowly rounded apex (Fig. 4b); female tergite VIII slightly sinuate apically on each side of disc (Fig. 4f), sternite VIII rounded apically, antecostal suture sinuate (Fig. 4g); spermatheca with large pear-shaped capsule, and long apical invagination, stem short and looped posteriorly, with slightly swollen apical part (Fig. 4e).

Distribution. Known from Quebec.
Collection and habitat data. Several adults were collected in November. Specimens from western Quebec were collected from dead and decaying black spruce (Picea mariana Mill. (BSP)) in a boreal forest dominated by black spruce. Diagnosis. Dinaraea piceana (habitus Fig. 5a) may be distinguished from congeners by the following combination of characters: body length 3.1-3.3 mm; head, pronotum and elytra matte with dense microsculpture; pronotum broadest in apical third and narrowest at base; elytra at suture slightly longer than pronotum, with dense punctation similar to that on pronotum; antennal articles 7-10 moderately transverse; male tergite VIII with four small apical teeth, all short and rounded (Fig. 5c); median lobe of aedeagus with straight and short tubus narrowly rounded apically and slightly produced ventrally (Fig. 5b); spermatheca with elongate pear-shaped capsule, and moderately long apical invagination, stem long, sinuate and looped posteriorly, with slightly larger apical part (Fig. 5e).
Distribution. Known from Quebec. Collection and habitat data. Most adults were collected in dead black spruce logs in boreal black spruce forests, and one was captured in an intercept trap in a boreal forest during July.
Description. Body length 3.3-3.7 mm; body dark brown with legs, antennae (at least basally), labial palpi and elytra yellowish-or reddish-brown, pronotum and abdomen lighter than head, sometimes entire body appears brown; head, pronotum and elytra slightly glossy, with dense microsculpture; abdominal microsculpture less dense and integument more glossy than that of pronotum and elytra; head about as broad as pronotum, genae slightly longer than eyes in dorsal view; pronotum broadest at middle, slightly transverse, usually longer than elytra at suture; elytra transverse, truncate posteriorly; abdomen subparallel; male tergite VIII with four small apical teeth, all short and rounded and some additional tooth-like subapical structures (Fig.  6c), sternite VIII produced apically (Fig. 6d); median lobe of aedeagus with short and straight venter of tubus and narrowly ventrally pointed apex (Fig. 6b); female tergite VIII concave basally and truncate apically (Fig. 6f), sternite VIII rounded apically and emarginated medially, antecostal suture slightly sinuate (Fig. 6g); spermatheca with short pear-shaped capsule, and small apical invagination, stem long, straight medially and looped posteriorly, with slightly swollen apical part (Fig. 6e).
Distribution. This Palaearctic species is adventive in North America . The earliest Canadian records are: Elora, Ontario in 1975 (DEBU); St. Andrews, New Brunswick in 1978 (DEBU); Fairview, Alberta in 1982 (DEBU). The first North American records are: Buffalo and New York before 1889 (Fauvel 1889); Davis, California before 1984 (Muona 1984). In Canada, the species is known from Newfoundland and Labrador, Nova Scotia, Prince Edward Island, New Brunswick, Quebec, Ontario, Alberta and Yukon Territory (Klimaszewski et al. 2007, 2010, 2011, 2012, Majka and Klimaszewski 2008, Webster et al. 2009). In the USA, it is reported from New York, Pennsylvania and California, but is likely more widely distributed in the northeastern states.
Collection and habitat data. In North America, this species is associated with soil and organic debris in agricultural fields and disturbed urban meadows. It is also found in marsh litter, in leaf litter in mixed forests, in compost, under bark of decaying spruce logs, amongst vegetation on a coastal sand dune, in litter in a cattail marsh, in Diagnosis. Dinaraea pacei (habitus Fig. 7a) may be distinguished from congeners by the following combination of characters: body length 2.2-2.5 mm; head, pronotum and elytra slightly glossy with dense microsculpture; pronotum broadest in apical third and narrowest at base; elytra at suture slightly longer than pronotum, with dense punctation similar to that on pronotum; antennal articles 7-10 moderately transverse; male tergite VIII with four minute apical teeth, all short and rounded, and scarcely visible (Fig. 7c); median lobe of aedeagus with strongly produced ventrally and narrowly rounded apically tubus (Fig. 7b); spermatheca with short pear-shaped capsule, and small apical invagination, stem short and looped posteriorly, with slightly swollen apical part (Fig. 7e). Dinaraea pacei is not distinguishable externally from Dinaraea subdepressa (Bernhauer), which was described from New Hampshire. However, the two species can be readily separated by the structures of the internal sac of the median lobe. The internal sac of D. subdepressa has two simple, straight, narrowly elongate sclerites (Fig. 13a), while the sclerites of D. pacei are complex as illustrated in Fig. 7b. The apical part of the median lobe is straight and slightly produced ventrally at the apex in D. subdepressa (Fig. 13a), while it is sinuate and strongly produced ventrally in D. pacei (Fig. 7b). The two species apparently represent sister taxa.
Description. Body length 2.2-2.5 mm; body dark brown with legs, antennae (at least basally), labial palpi and elytra slightly paler and appearing reddish-brown, pronotum and abdomen lighter than head, sometimes entire body appears brown; head, pronotum and elytra slightly glossy, with dense microsculpture; abdominal microsculpture less dense and integument more glossy than pronotum and elytra; head about as large and broad as pronotum, genae slightly longer than eyes in dorsal view; pronotum broadest in apical third, slightly transverse, usually shorter than elytra at suture; elytra transverse, truncate posteriorly; abdomen subparallel; male tergite VIII with four minute apical teeth, all short and rounded, and scarcely visible (Fig. 7c); sternite VIII arcuate apically (Fig. 7d); median lobe of aedeagus with short and sinuate tubus and narrowly dorsally pointed apex (Fig. 7b); female tergite VIII concave basally and truncate apically (Fig. 7f), sternite VIII rounded apically and emarginated medially, antecostal suture slightly sinuate (Fig. 7g); spermatheca with short pear-shaped capsule, and small apical invagination, stem short and looped posteriorly, with slightly swollen apical part (Fig. 7e).
Distribution. This species was previously recorded only from southwest Labrador ). Here, we provide new distribution data from British Columbia, New Brunswick, Quebec and Alaska.
Collection and habitat data. Adults in Newfoundland and Labrador were collected from June to August using pitfall traps and flight intercept traps in various coniferous forest types, and one specimen was collected under the bark of a dead red pine . In British Columbia, adults were caught in July and September in emergence traps attached to the trunks of lodgepole pine (Pinus contorta Dougl. ex Loud. latifolia Engelm.) infested by mountain pine beetle (Dendroctonus ponderosae Hopkins). In New Brunswick, adults were found: under the bark of large fallen spruce in an old-growth eastern white cedar swamp; under tight bark of American elm; in a silver maple forest; in fleshy polypore fungi at the base of a dead standing Populus sp. in a wet alder swamp; and in a group of Pholiota sp. at the base of a dead Populus sp. in a mixed forest. In Quebec, adults were found in dead black spruce in a black spruce forest. Adults were also captured in Lindgren funnel traps deployed in an old-growth white spruce (Picea glauca (Moench) Voss) and balsam fir forest, an old mixed forest with red and white spruce, red and white pine (Pinus strobus L.), and a rich Appalachian hardwood forest with some conifers. Adults were collected from March to September. Dinaraea borealis Lohse, in Lohse et al. 1990: 198 (based on a single female description). HOLOTYPE (female): CANADA, QUEBEC, Gt. Whale River, 30.VI.1949, J.R. Vockeroth, No. 20341 (CNC). Holotype examined.
Diagnosis. Dinaraea borealis (habitus Fig. 8a) may be distinguished from congeners by the following combination of characters: body length 2.8-3.0 mm; head, pronotum and elytra slightly glossy with dense microsculpture; pronotum broadest in apical third and narrowest at base; elytra at suture as long as pronotum, with dense punctation similar to that on pronotum; antennal articles 7-10 moderately transverse; male tergite VIII without apical teeth (Fig. 8c); median lobe of aedeagus with straight, long tubus pointed at apex and slightly pointed ventrally (Fig. 8b); spermatheca elongate with pear-shaped capsule, and long apical invagination, stem long and looped posteriorly, with strongly swollen apical part (Fig. 8e).
Description. Body length 2.8-3.0 mm; body variable in colour, either entirely black with brown or reddish appendages and part of elytra, or dark brown with legs,  Etymology. Curtipenis means 'possessing a short penis', in allusion to the short tubus of the median lobe of the aedeagus in this species.
Diagnosis. Dinaraea curtipenis (habitus Fig. 9a) may be distinguished from congeners by the following combination of characters: body length 3.0 mm; head, pronotum and elytra slightly glossy with dense microsculpture and bluish tinge; pronotum broadest in the middle and narrowest at base; elytra at suture as long as pronotum, with dense punctation similar to that on pronotum; antennal articles 7-10 moderately transverse; male tergite VIII without apical teeth (Fig. 9c); median lobe of aedeagus with straight, short tubus pointed at apex and slightly pointed ventrally (Fig. 9d), internal sac with distinct sclerites (Fig. 9b). Dinaraea curtipenis is superficially similar to D. subdipressa but can be distinguished from it by lacking a strongly depressed posterior edge of pronotum, while it is strongly depressed at the posterior angle of the disc and medially at base forming a groove in the latter species; and it has more elongate bulbus of median lobe of aedeagus with additional dorsal and ventral sclerotized structures (Fig. 9b).
Description. Body length 3.0 mm; body dark brown with reddish-brown tarsi, tibia and bases of antennae; head, pronotum and elytra slightly glossy, elytra more so, with dense microsculpture; abdominal microsculpture less dense and integument more glossy than that of pronotum and elytra; head large, as broad as pronotum, genae slightly longer than eyes in dorsal view; pronotum broadest in middle, slightly transverse, usually as long as elytra at suture; elytra transverse, truncate posteriorly; abdomen subparallel; male tergite VIII truncate apically and without apical teeth (Fig.  9c); sternite VIII rounded apically (Fig. 9d); median lobe of aedeagus with short and straight tubus with apex pointed ventrally (Fig. 9b). Female undescribed. FEMALE. We have several females from New Brunswick localities found without male association that may belong to this species but because they are very similar externally and have similar genitalia to those of D. pacei we hesitate to formally associate them with D. curtipenis. These females have a more coarsely punctate pronotum than those of D. pacei but have similar rounded posterior angles of pronotum. We anticipate that this problem will be solved by finding females associated with males at the same locality. Distribution. This species is known only from New Brunswick. Collection and habitat data. The holotype was collected from under the bark of a conifer log in a mixed forest. Other individuals were captured in Lindgren funnel traps in an old red oak forest, an old-growth eastern white cedar forest, an old-growth white spruce and balsam fir forest, and old red pine forests. The paratype was collected from a well-decayed and fungus-covered log in a mixed forest. Adults were collected during May and July. Etymology. Longipenis means 'possessing a long penis', in allusion to the long tubus of the median lobe of the aedeagus in this species.

Dinaraea longipenis
Diagnosis. Dinaraea longipenis (habitus Fig. 10a) may be distinguished from congeners by the following combination of characters: body length 3.5 mm; head, pronotum and elytra slightly glossy with dense microsculpture; pronotum broadest in the middle and narrowest at base; elytra at suture shorter than pronotum, with dense punctation similar to that on pronotum; antennal articles 7-10 strongly transverse; male tergite VIII without apical teeth (Fig. 10c); median lobe of aedeagus with straight, long tubus rounded at apex and slightly pointed ventrally (Fig. 10b), internal sac with thin sclerites (Fig. 10b). FEMALE: We have one female from New Brunswick found without male association that may belong to this species. This female is externally very similar to the male of D. longipenis but the spermatheca and tergite and sternite VIII are indistinguishable from that of D. piceana. We need more specimens of this species, with male and female association, to confirm and formally describe the female of this species.
Description. Body length 3.5 mm; body dark black with light reddish-brown tarsi, tibia and bases of antennae; head, pronotum and elytra slightly glossy, the elytra more so, with dense microsculpture; abdominal microsculpture less dense and integument more glossy than that of pronotum and elytra; head large, about as broad as pronotum, genae slightly longer than eyes in dorsal view; pronotum broadest in middle, slightly transverse, longer than elytra at suture; elytra transverse, truncate posteriorly; abdomen arcuate laterally; male tergite VIII truncate apically and without apical teeth, slightly angular laterally at apical margin (Fig.  10c), sternite VIII rounded apically (Fig. 10d); median lobe of aedeagus with long and straight tubus with apex rounded and pointed ventrally (Fig. 10b). Female undescribed (see above).
Distribution. This species is known only from New Brunswick.
Collection and habitat data. The holotype was captured during May in a Carex marsh by treading emergent vegetation. The paratype was collected from a well-rotted log in a hardwood forest. son, Katie Burgess, Marie-Andrée Giguère, Cory Hughes, Colin MacKay, Michelle Roy, and Vincent Webster for technical assistance and collecting specimens; Natural Resources Canada -Canadian Forest Service, Canadian Food Inspection Agency, and USDA APHIS for funding the study on early detection of invasive cerambycids, which provided many specimens collected in Lindgren funnel traps; the Canadian Wildlife Service for funding insect surveys at the Shepody National Wildlife Area; the New Brunswick Wildlife Trust Fund for funding various insect surveys over the past 7 years; the Meduxnekeag River Association for permission to sample beetles at the Meduxnekeag Valley Nature Preserve (which includes the Bell Forest); and New Brunswick Department of Natural Resources (Fish and Wildlife Branch) for issuing permits for sampling in the Protected Natural Areas and for providing logistical support. Survey work in the Jacquet River Gorge Protected Natural Area and the Caledonia Gorge Protected Area was organized through the New Brunswick Museum with external funding from the New Brunswick Environmental Trust Fund, Salamander Foundation, and the New Brunswick Wildlife Trust Fund. We appreciate the assistance of James Hammond (NoFC) in drafting the distribution maps, Pamela Cheers (LFC) in editing and Diane Paquet (LFC) in formatting the manuscript. This project was financed by the Canadian Forest Service, Natural Resources Canada.


extfieldsofstudy: Medicine Biology

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Identification of novel antimicrobial resistance genes from microbiota on retail spinach

Background Drug resistance genes and their mobile genetic elements are frequently identified from environmental saprophytic organisms. It is widely accepted that the use of antibiotics in animal husbandry selects for drug resistant microorganisms, which are then spread from the farm environment to humans through the consumption of contaminated food products. We wished to identify novel drug resistance genes from microbial communities on retail food products. Here, we chose to study the microbial communities on retail spinach because it is commonly eaten raw and has previously been associated with outbreaks of bacterial infections. Results We created metagenomic plasmid libraries from microbiota isolated from retail spinach samples. We identified five unique plasmids that increased resistance to antimicrobial drugs in the E. coli host. These plasmids were identified in E. coli that grew on plates that contained ampicillin (pAMP), aztreonam (pAZT), ciprofloxacin (pCIP), trimethoprim (pTRM), and trimethoprim-sulfamethoxazole (pSXT). We identified open reading frames with similarity to known classes of drug resistance genes in the DNA inserts of all 5 plasmids. These drug resistance genes conferred resistance to fluoroquinolones, cephalosporins, and trimethoprim, which are classes of antimicrobial drugs frequently used to treat human Gram negative bacterial infections. These results show that novel drug resistance genes are found in microbiota on retail produce items. Conclusions Here we show that microbiota of retail spinach contains DNA sequences previously unidentified as conferring antibiotic resistance. Many of these novel sequences show similarity to genes found in species of bacteria, which have previously been identified as commensal or saprophytic bacteria found on plants. We showed that these resistance genes are capable of conferring clinically relevant levels of resistance to antimicrobial agents. Food saprophytes may serve as an important reservoir for new drug-resistance determinants in human pathogens.

Background
The spread of antimicrobial resistance genes has made previously manageable bacterial infections increasingly more difficult to treat. In addition, there has been a gradual decline in the development of new antimicrobial drugs, especially against Gram negative bacterial pathogens. The identification of genes in Gram negative bacteria that confer resistance to cephalosporins, carbapenems, and fluoroquinolones has created fears that we are returning to the pre-antibiotic era [1]. These multidrug-resistant infections often occur in hospitals and are frequently caused by species belonging to the normal microbiota of the human host [1,2]. This suggests that the microbiota of the patients themselves is the reservoir for many of the organisms that cause hospital acquired infections. Furthermore, recent work has demonstrated that the intestinal microbiota of humans and food animals are a reservoir of drug resistance genes [3]. Consequently, a better understanding of how drug resistance genes enter the human microbiota is imperative to better prevent drug resistant infections.
Drug resistance genes and their mobile genetic elements are frequently identified from environmental saprophytic organisms. These include samples taken from soil, water, and wild animals [4][5][6]. Additionally, these genes have been identified in environmental samples from ancient and pristine environmentssamples that have never been exposed to human activity [4,5,7]. Due to the great diversity of antibiotic resistance genes found in environment, it has been hypothesized that environmental microbes serve as a reservoir of drug resistance genes and that a few then enter human pathogens [4,5]. These drug resistance genes are spread between bacteria via mobile genetic elements, such as plasmids, transposons, and integrons [4][5][6]8]. The detection of mobile genetic elements and drug resistance genes in the environment has led to numerous studies and policies to address the effects of environmental exposure to antimicrobial agents on human pathogens [9,10].
It is widely accepted that the use of antibiotics in animal husbandry selects for drug resistant microorganisms, which are then spread from the farm environment to humans through the consumption of contaminated food products [11]. Numerous studies of bacterial pathogens in food products, such as Campylobacter and Salmonella, have demonstrated that the use of antimicrobial drugs in agriculture can result in drug resistant infections in humans [9][10][11][12][13]. However, the majority of studies have been limited to species of zoonotic pathogens that cause foodborne disease and these studies are frequently done as part of national surveillance programs for food safety. Species of bacteria that are not usually considered foodborne pathogens, but nonetheless are found in both the human and food product microbiota, are usually not included in studies of drug resistant bacteria in retail food products. Studies of microbiota of animals demonstrated that commensal organisms are a reservoir of antimicrobial drug resistance genes [3]. These studies include the identification of antimicrobial resistance genes from animal feces including chickens and cows [3,[14][15][16]. Animal manure is frequently used as fertilizer in agriculture and may contribute to the spread of drug resistance genes. The spread of drug resistance genes by commensal bacteria on food products is an area that requires further study [11].
Produce items, which are frequently eaten raw, are one way consumers are exposed to microbiota on retail food products [17]. Previous work has shown that the normal microbiota of retail produce items harbors clinically relevant drug resistance genes [18]. However, previous studies have relied on PCR based methods to identify known drug resistance genes, which limits the number and types of drug resistance that could potentially be identified. Other studies used functional metagenomic libraries to identify novel antimicrobial resistance genes from environmental samples in a sequence independent manner [19,20]. We wanted to apply this sequence independent approach to investigate the presence of antimicrobial resistance genes on retail spinach. We chose to study the microbial communities on retail spinach because it is commonly eaten raw and has previously been associated with outbreaks of bacterial infections [21].
To do this, we made two metagenomic plasmid libraries with DNA isolated from the microbiota of retail spinach. One library was made from a cultured sample of spinach microbiota while the other was made in a culture independent manner. We then screened these libraries for their ability to confer resistance to antibiotics to an E. coli host.

Results and discussion
Isolation of antibiotic resistant clones The first plasmid library, which was constructed from a cultured sample, contained 160 Mb of inserted DNA. The second library, which was constructed from an uncultured sample, contained 140 Mb of inserted DNA. We first constructed a cultured library because we wished to enrich for microbial DNA to increase the chances of cloning DNA sequences that contained drug-resistance genes. We constructed a library from an uncultured sample because we wanted to identify potential drug-resistance genes from bacterial organisms that cannot be cultivated in artificial medium. From the cultivated library, we isolated four different antimicrobial resistance-conferring clones. From the uncultivated library, we isolated one additional antimicrobial resistance-conferring clone.
The mean size of the plasmid DNA inserts in both libraries was two Kb. We identified five unique plasmids that conferred increased drug resistance (minimum inhibitory concentration or MIC) to the host E. coli. Each plasmid was named after the antimicrobial agent to which it conferred resistance (Table 1). These plasmids were identified in E. coli that grew on plates that contained ampicillin (pAMP), aztreonam (pAZT), ciprofloxacin (pCIP), trimethoprim (pTRM), and trimethoprim-sulfamethoxazole (pSXT). The plasmids pAMP, pAZT, pCIP, and pTRM were isolated from the library made from a cultured sample. The plasmid pSXT was isolated from the library made from an uncultured sample.

Identification of antibiotic resistance genes and phylogenetic analysis
We identified open reading frames with similarity to known classes of drug resistance genes in DNA inserts of all 5 plasmids. They are summarized in Figure 1 and Table 2.
In pAZT we identified a sequence with 94% identity at the nucleotide level to a gene that encodes penicillinbinding protein 1A identified in Bacillus subtilis subsp. Spizizenii [GenBank, gb|CP002905.1|]. The expression of altered penicillin binding proteins are known to confer resistance to beta-lactam and cephalosporin antibiotics in various clinically important pathogens [22]. However, the ability of this sequence to confer clinically relevant levels of cephalosporin resistance or an ESBL phenotype has not been previously reported.
In pAMP we identified a beta-lactamase gene with 71% identity to the ERP-1 gene that encodes a class A extended spectrum beta-lactamase found in Erwinia persicin [GenBank, gb|AY077733.1|] [23]. When transformed into an E. coli host, ERP-1 was reported to increase resistance to penicillins and cephalosporins, including piperacillin, cefotaxime, and ceftazidime [23]. Surprisingly, we found that pAMP did not increase the MIC of the host E. coli to piperacillin, ceftazidime, or cefotaxime. Also, pAMP did not increase the MIC of this host strain to any tested cephalosporin, monobactam, or carbapenem (cefepime, aztreonam, or imipenem). However, this isolate tested positive for the presence of a beta-lactamase by the nitrocefin assay. These results suggest that the novel sequence we identified in pAMP is distinct from ERP-1 in terms of the spectrum of drug-resistance phenotype it encodes. However, it is also possible that these observations are artifacts due to poor expression of the gene in a heterologous host.
Surprisingly, when the pCIP DNA sequence was submitted to BlastN, only two other sequences in the NCBI non-redundant nucleotide database were identified. The sequences were part of whole genome sequences of Exiguobacterium antarcticum and Exiguobacterium sibiricum. These species were identified in a frozen Antarctic lake and a core sample of the Siberian tundra [24,25]. When MICs in the presence of the plasmid with the metagenomic DNA insert compared to the empty vector (pSMART or 1B) are shown. In accordance with manufactures recommendations, less than a 4-fold increase was considered to be within the margin of error.
submitted to BlastP, a fluoroquinolone resistance protein from Oceanobacillus sp. Ndiop was identified. This quinolone resistance protein was a predicted pentapeptide repeat protein (PRP) [GenBank, ref|ZP_10910075.1|]. One known class of plasmid-mediated quinolone resistance conferring sequences is called QNR [26]. QNRs are pentapeptide repeat proteins and have been associated with extended spectrum beta-lactamases [26]. In addition to the PRP, the pCIP DNA insert contained a second open reading frame that showed similarity to a beta-lactamase domain containing protein (Figure 1). However, pCIP did not increase the MIC of the host E. coli to any of the tested betalactam antibiotics (ampicillin and piperacillin), cephalosporin antibiotics (ceftazidime, cefotaxime, cefepime), or carbapenem antibiotics (imipenem). Additionally, pCIP did not test positive for beta-lactamase production by the nitrocefin assay. This indicated that the predicted betalactamase is either not expressed or does not function as a beta-lactamase. We subcloned the PRP sequence in pCIP (pPRP:1B) in order to confirm that the predicted PRP was able to confer resistance to ciprofloxacin. As compared to the empty vector, the pPRP:1B increased the MIC of ciprofloxacin of the host E. coli 7 fold (.016 to .125) and the MIC of levofloxacin 5 fold (.047 to .25 ug/ml). The MIC conferred by pPRP:1B to the host E. coli is consistent with previously reported MICs from other QNR sequences found in human pathogens [26].
We found that the pTRM DNA insert has 84% identity to a region of the Erwinia pyrifoliae DSM 12163 complete genome [GenBank, emb|FN392235.1]. This region contains the folA gene, which encodes a dihydrofolate reductase (DHFR). DHFR is the target of trimethoprim [27]. The expression of a DHFR that is not susceptible to trimethoprim is a well-known mechanism of resistance [27]. However, the acquisition of the folA gene from Erwinia pyrifoliae DSM 12163 has not previously been shown to confer resistance to trimethoprim.
Similarly, the DNA insert from pSXT has 88% identity to a region of the Pseudomonas fluorescens SBW25 complete genome [28]. This region also encodes a predicted dihydrofolate reductase. This sequence from Pseudomonas fluorescens SBW25 has not previously been shown to confer resistance to trimethoprim or trimethoprim sulfamethoxazole.

Conclusions
Here we show that microbiota of retail spinach contains previously unidentified antibiotic resistance-conferring genes and that functional metagenomic libraries can be used to screen retail food products for drug resistance genes in a sequence independent manner. Furthermore, Figure 1 Diagram of open reading frames and DNA insert size. Open reading frame labels correspond to those in Table 2. Broken arrows correspond to truncated open reading frames. Open reading frames marked with a * show similarity to known drug resistance genes. Figure not drawn to scale.
due to the limited amount of DNA that can be cloned into a plasmid library and the requirement that the drug resistance gene be expressed in a heterologous host, it is likely we only identified a fraction of drug resistance conferring genes present in our spinach samples.
Although none was identical in DNA sequence, many of these novel sequences show sequence similarity to genes found in species of bacteria that have previously been identified as commensal or saprophytic bacteria found on plants [23,28]. This suggests the sequences we identified are not the result of contamination from animals or humans. We showed that these resistance genes are capable of conferring clinically relevant levels of resistance to commonly used classes of antimicrobial agents, including cephalosporins and fluoroquinolones.
The novel antimicrobial resistance genes we identified include beta-lactamases, a pentapeptide repeat protein, a penicillin binding protein, and putative dihydrofolate reductase genes. These types of resistance mechanisms are some of the most common and clinically problematic mechanisms of drug resistance found in pathogens [27]. We do not know at this time if these genes will become clinically important, and one limitation of this study is that we did not analyze these genes for their potential for horizontal transfer to human pathogens. Recent functional genomics analysis of environmental soil samples has not only identified drug-resistance genes with identical nucleotide sequences from human pathogens, but also mobile gene sequences providing evidence for possible horizontal gene transfers [29]. Further studies using sequence independent methods to identify antimicrobial resistance genes from retail food products should be done to better understand the role of saprophytes as a reservoir for new drug-resistance genes.

Metagenome plasmid library construction and screening
Two metagenomic plasmid libraries of spinach microbiota were constructed. One was based on DNA extracted from cultured bacteria and the other was based on DNA extracted from uncultured spinach wash. The metagenomic DNA used to create the first library was obtained by washing twenty five grams of bagged "baby spinach" in PBS. A description of the spinach used to create the library has been previously published [18]. Briefly, the spinach was purchased from a supermarket located in Berkeley, California in 2007. They included organic as well as non-organic spinach. One milliliter of the PBS wash was then used to inoculate 50 ml TSB. This culture was grown at 37°C with shaking overnight. The culture was then centrifuged at 10,000 x g for 10 minutes. DNA was extracted from the resulting pellet by the phenol chloroform method. The metagenomic DNA used to create the second library was obtained by washing six bags of "baby spinach" in two liters of PBS. Six different brands of spinach were purchased from three retailers located in Berkeley, California in 2011. The spinach was incubated in PBS at room temperature for two hours. The resulting wash was then filtered through sterilized cheesecloth and a sterilized coffee filter to remove spinach debris. The filtered wash was then centrifuged at 10,000 x g for 20 minutes. DNA was extracted directly from the resulting pellet with the Gnome DNA isolation Kit, MP Biomedical.
Two plasmid libraries with metagenomic DNA inserts were constructed in pSMART-LC kan vector in the E. coli host, E. Cloni (Lucigen corp., Middleton WI). The pSMART vector confers resistance to kanamycin and has transcriptional terminators flanking the cloning sites. Consequently, transcription of the cloned sequences requires a native promoter.
E. coli clones containing the two plasmid libraries were then screened for resistance to antimicrobial agents on Mueller Hinton agar plates containing one of the following 16 antimicrobial agents: ampicillin, carbenicillin, ticarcillin, amoxicillin/clavulanic acid, ticarcillin/clavulanic acid, cefotaxime, ceftazidime, aztreonam, meropenem, gentamicin, nalidixic acid, ciprofloxacin, trimethoprim, trimethoprimsulfamethoxazole, chloramphenicol, or tetracycline. The phenotype of resistance to antimicrobial agents was confirmed by retransforming the recombinant plasmid into E. Cloni. Growth on Muller Hinton agar containing kanamycin was used as a positive control for transformation. The acquisition of drug resistance from the transformation of the plasmid was demonstrated by growth on Mueller Hinton agar containing the corresponding antimicrobial agent. We used the empty vector, pSMART, in the E. coli host as a negative control for antibiotic stability.

Antimicrobial susceptibility testing
The MIC of each E. coli clone was determined by Etest (Biomerieux, France) according to manufactures recommendations. All Etests were repeated in at least two independent experiments. ATCC 29522, ATCC 700603, and ATCC 35218 were used for control as recommended by the manufacturer. In accordance with manufactures recommendations, less than a fourfold difference in MIC was considered to be with in the expected margin of error for this test.

Nitrocefin test
A colony was spotted onto a sterile Petri dish and then covered with nitrocefin, as previously described [30]. E.
Cloni containing the empty vector (pSMART) was used as a negative control.
Sequencing and data analysis: The sequence of the DNA insert in the resistance conferring plasmid was determined by primer walking at the University of California, Berkeley sequencing facility. The sequences were assembled with Geneious Version 5.6, (Biomatters, New Zealand). The insert sequences were then submitted to ORFinder and the BLAST suit of programs at NCBI [31]. The nucleotide sequences of the insert from each plasmid have been deposited in Genbank with the following accession numbers: pAMP: KF791056, pAZT: KF791057, pCIP: KF791058, pTRM: KF791059, pSXT: KF791060.


Detection of Quantitative Trait Loci (QTL) Related to Grilsing and Late Sexual Maturation in Atlantic Salmon (Salmo salar)

In Atlantic salmon aquaculture, early sexual maturation represents a major problem for producers. This is especially true for grilse, which mature after one sea winter before reaching a desirable harvest weight, rather than after two sea winters. Salmon maturing as grilse have a much lower market value than later maturing individuals. For this reason, most companies desire fish that grow fast and mature late. Marker-assisted selection has the potential to improve the efficiency of selection against early maturation and for late sexual maturation; however, studies identifying age of sexual maturation-related genetic markers are lacking for Atlantic salmon. Therefore, we used a 6.5K single-nucleotide polymorphism (SNP) array to genotype five families from the Mainstream Canada broodstock program and search for SNPs associated with early (grilsing) or late sexual maturation. There were 529 SNP loci that were variable across all five families, and this was the set that was used for quantitative trait loci (QTL) analysis. GridQTL identified two chromosomes, Ssa10 and Ssa21, containing QTL related to grilsing. In contrast, only one QTL, on Ssa18, was found linked to late maturation in Atlantic salmon. Our previous work on these five families did not identify genome-wide significant growth-related QTL on Ssa10, Ssa21, or Ssa18. Therefore, taken together, these results suggest that both grilsing and late sexual maturation are controlled independently of one another and also from growth-related traits. The identification of genomic regions associated with grilsing or late sexual maturation provide an opportunity to incorporate this information into selective breeding programs that will enhance Atlantic salmon farming.

Introduction
The Atlantic salmon (Salmo salar) exhibits a great deal of variability in age and size at sexual maturation. This variation is observed both between and within strains and year classes (Garcia de Leaniz et al. 2007;Taranger et al. 2010). The timing of sexual maturation is controlled by a complex process that involves genetic and environmental components (Thorpe and Metcalfe 1998). Internal factors, such as age and lipid reserves, together with external factors, like light abundance, temperature, and food intake, seem to have an impact on the initiation of sexual maturation (Gardner 1976;Herbinger and Friars 1992;Simpson 1992). The interactions of these factors result in the enormous variability in the age of sexual maturation, especially in male Atlantic salmon, which are able to reach maturity at 1 to 7 years of age (Simpson 1992). There is also a genetic component to age at sexual maturation in Atlantic salmon, and estimates of heritabilities (h 2 ) for this trait in Atlantic salmon range widely from 0.09-0.17 Wild et al. 1994;Gjedrem 2000) to 0.39 (Gjerde and Gjedrem 1984) to 0.48 (Gjerde 1984). These observations strongly indicate that age at sexual maturity in Atlantic salmon is a heritable trait. Thus, it should be possible to select for Atlantic salmon that do not become sexually mature as grilse and also for late sexually maturing fish.
In salmon aquaculture, early sexual maturation can be a major economic problem. This is particularly costly in terms of feed and cage space if fish mature as grilse (i.e., after 1 year in a sea cage; Gjedrem 2000). Salmon maturing as grilse have a much lower market value than later maturing individuals (Saunders et al. 1983). The maturation process is energetically expensive, and this is reflected in a decrease in growth rate, lower meat quality, and increased mortality through susceptibility to pathogens (Gjerde 1984;Thorpe 1994). Late-maturing salmon are desired in commercial operations, and to reduce the frequency of grilsing, early maturing Atlantic salmon are discarded as potential breeding candidates (Gjedrem 2012). Selection for the economically important production traits of fast growth and late sexual maturation has been considered problematic as it has been suggested that there is a correlation between the phenotypes of fast growth and early sexual maturation (Thorpe et al. 1983).
Marker-assisted selection has the potential to improve the efficiency of selection for traits such as age of sexual maturation and growth in Atlantic salmon breeding. Genomic regions linked to sexual maturation have been identified in other salmonid species such as rainbow trout and Arctic charr (Easton et al. 2011;Haidle et al. 2008;Küttner et al. 2011;Martyniuk et al. 2003;Moghadam et al. 2007). However, such studies in Atlantic salmon are lacking. The identification of genetic markers independently related to either early or late sexual maturation, especially in male Atlantic salmon, would enable the implementation of selective breeding based on improved genetic selection practices by identifying animals with favorable genotypes. We previously identified QTL for growth at different stages of the production cycle in five families from the Mainstream Canada broodstock (Gutierrez et al. 2012). Here we report QTL for grilsing and late sexual maturation in the same families and show that the genomic locations of these QTL are independent of one another as well as the QTL associated with growth.

Mapping Families and Phenotype Data
Families were part of a commercial broodstock program developed by Mainstream Canada and based on the Mowi strain of Atlantic salmon, which was derived from a breeding program established using Norwegian populations (Gjedrem et al. 1991). In November/December 2005, 130 single-pair mating families were established. At the fry stage (February 2006), 120 offspring from each family were pooled (15,600 fish in total) and grown communally at the Oceans Farms Hatchery, Vancouver Island. In September/October 2006, 5,000 of the fish were passive integrated transponder (PIT)tagged, and phenotypic measurements were taken until early 2009. Maturation times in Atlantic salmon were classified as: precocious (≤12 months of age), grilse (36 months of age, at first sea winter (SW)), normally maturing (48 to 60 months of age at second SW or third SW), and late-maturing fish (>60 months) (Gjedrem 2000;Taranger et al. 2010). Sex was recorded during the confirmation of maturation status. The sex of an individual in the late-maturing group could not be ascertained phenotypically; therefore, we predicted the sex of the late-maturing Atlantic salmon using a polymerase chain reaction test, developed by Eisbrenner et al. (2013) that amplifies two exons of the sdY gene, which has been shown to be the sex-determining gene in rainbow trout and probably other members of the Salmoninae (Yano et al. 2012(Yano et al. , 2013. It should be noted that the precocious Atlantic salmon that matured before 1 year of age were discarded during a standard grading procedure, and so no samples were available from this group.

DNA Extraction and Parental Assignments
These procedures were as described by Gutierrez et al. (2012). Briefly, DNA was extracted from the adipose fins of the PITtagged progeny and the parents used to produce the 130 families (Withler et al. 2005). The DNA was then genotyped using eight microsatellite markers, and parental assignment was carried out as described by Withler et al. (2007).

SNP Array and Linkage Mapping
The single-nucleotide polymorphism (SNP) data used for this analysis have been described previously (Gutierrez et al. 2012). Five families were selected for SNP genotyping at CIGENE, Norwegian University of Life Sciences, Ås, Norway, using an Atlantic salmon 6.5K Illumina iSelect SNP-array (Kent et al. 2009). Analyses were based on an Atlantic salmon linkage map, which contains ∼5,650 SNP markers and was constructed using genotyping data from 143 families comprising 3,297 fish ). This map contains 29 linkage groups, which were assigned to their specific chromosome number according to the nomenclature established by Phillips et al. (2009). All non-informative markers were removed from the datasets, and subsequently, independent male and female linkage maps were manually created for each family and adjusted to the distances on the SNP map of Lien et al. (2011). Given the differences between the sets of informative markers present in each family, for this study, we restricted the analysis to the 529 markers which are informative in all of the five families. These markers are distributed across all 29 Atlantic salmon chromosomes. We have previously commented on some of the possible reasons why only ∼10% of the markers were variable in all of the five families (Gutierrez et al. 2012). It may reflect the starting population for the Mainstream Canada broodstock or past selection (e.g., primarily for growth in terms of days in salt water to reach 5 kg), or it may reflect the nature of biallelic SNPs. We note that many QTL studies in salmonids have traditionally used microsatellite markers, and these studies tend to have employed fewer variable markers and also fewer families. Even when SNPs were used as the genetic marker, the overall number tested for QTL analysis has been less than 350, and not all of these were variable in each family. Therefore, we consider this study to be robust in terms of what is currently available in the salmonid aquaculture literature.

QTL Analyses
QTL analyses were performed using a regression-interval mapping method available through the GridQTL portlet (Seaton et al. 2006). GridQTL is a portlet environment (available at http:// www.gridqtl.org.uk/) that allows the analysis of extensive datasets. The datasets were analyzed as sib-pairs using the combined genotypic data from the 529 informative markers shared by all families. For the grilsing analysis, given the absence of female grilse, only the genotypic information from males was used. The phenotypic data were scored as a binary trait, i.e., male fish recorded as grilse were scored 0, and male fish recorded as maturing at second or third SW were scored as 1. The same procedure was used for the analysis of late sexual maturation, but both male and female fish were used in this case. Atlantic salmon that did not show evidence of gonadal development at 60 months were considered "late-maturing" and scored as 0. The results were illustrated using the female map as it provides a greater resolution than the male genetic map.
The percentage of phenotypic variance (PEV) explained by the QTL in the sib-pair analysis was calculated as a proportion of the phenotypic variance obtained from the observations. Fstatistic values were calculated at 1 cM intervals on each chromosome for each analytical approach to yield the most likely position of the QTL. Empirical chromosome-wide significance thresholds were determined by permutation tests (Churchill and Doerge 1994). The 10,000 permutations were performed at the chromosome-wide level in order to establish the F-value thresholds for a p<0.05 and p<0.01. F-critical values corresponding to 0.05>p>0.01 were considered as "suggestive" QTL, whereas those corresponding to p<0.01 were considered "significant" QTL. Those chromosomes which contained QTL that were found to be "significant" were tested for their significance at a genome-wide level of p<0.05 by performing a 1,000 permutation test.

QTL Analysis for Male Grilsing
As described previously (Gutierrez et al. 2012), five families were chosen from the 2005 year class of the Mainstream Canada selective breeding program for analysis. Each family contained from five to eight male grilse. No female grilse were observed in any of these families ( Table 1). As shown in Fig. 1 and Table 2, QTL analyses restricted to male grilsing revealed the presence of two statistically meaningful QTL, one on chromosome 10 (Ssa10) and the other on chromosome 21 (Ssa21). The QTL on Ssa21 was considered significant (p<0.01), whereas the other on Ssa10 only reached the suggestive level of significance (0.05>p>0.01). Neither of these QTL reached genome-wide significance, which is in keeping with the relatively small sample size as well as the rather low h 2 estimates in Atlantic salmon for age at sexual maturation (Gjerde 1984;Gjerde et al. 1994;Wild et al. 1994). The PEV explained by these QTL was 16.7% and 25% in Ssa10 and Ssa21, respectively.

QTL Analysis of Late Sexual Maturation
From the 195 individuals used in the analysis of late sexual maturation, 40 were fish that did not show gonadal development after their third SW. The sex of the individuals in this latter group was determined based on the presence of two of the four exons of the sdY gene (Eisbrenner et al. 2013). Seventeen of the late-maturing 40 fish were male and 23 were female (Table 1). The GridQTL analysis revealed the presence of only one statistically significant (p<0.01) QTL on chromosome 18 (Ssa18), which did not reach genome-wide significance, even though it was the only QTL found for this particular trait (Fig. 2). This QTL explained 14.7 % of the PEV associated with late sexual maturation in these Atlantic salmon families.

Discussion
Previous studies performed in rainbow trout and Arctic charr have shown an apparent link between QTL for sexual maturation and growth (Haidle et al. 2008;Martyniuk et al. 2003;Moghadam et al. 2007); however, our results indicate a different scenario for Atlantic salmon in the Mainstream Canada breeding program. Here we only detected two QTL associated with male grilsing in Atlantic salmon, on Ssa10 and Ssa21. Previously using the same families, Gutierrez et al. (2012) found genome-wide significant growth-related QTL on chromosomes Ssa02, Ssa07, Ssa13, Ssa09, Ssa17, and Ssa26. Although Gutierrez et al. (2012) found some suggestive evidence for growth-related QTL on Ssa10 and Ssa21, neither reached genome-wide significance, and they mapped to different regions of the chromosomes compared with the grilsing QTL. Moreover, the results of QTL analyses for growth and sexual maturation in the Mainstream Canada broodstock families indicate that sexual maturation is controlled by fewer genomic regions than growth in Atlantic salmon. Therefore, it should be possible to tease apart these traits genetically and so select for each independently using specific genetic markers. A different situation was observed for the QTL for late sexual maturation detected on Ssa18. To date, no growthrelated QTL have been reported on this chromosome in Atlantic salmon or on homologous chromosomes in other salmonids (Gutierrez et al. 2012).
Correlations between the phenotypes of growth (as estimated by body weight) and early sexual maturation have been reported in Atlantic salmon (Gjerde 1984;Gjerde et al. 1994;Wild et al. 1994). Moreover, considerable genetic variation in age at sexual maturity has been described in farmed fish species. For example, in Atlantic salmon h 2 estimates range from 0.09 to 0.17 (Gjerde 1984;Gjerde et al. 1994;Wild et al. 1994) to 0.39 (Gjerde and Gjedrem 1984) to 0.48 (Gjerde 1984) whereas in rainbow trout h 2 estimates range from 0.12 to 0.35 (Gjerde and Schaeffer 1989;Kause et al. 2003). Nevertheless, selective breeding programs have been effective in increasing body size while also controlling undesired early sexual maturity in farmed fish (Gjedrem 2000), giving clues that these traits are influenced by different genes. Our previous results showed that growth-related QTL were spread across the Atlantic salmon genome (Gutierrez et al. 2012); however, none of the QTL associated with grilsing or late sexual maturation were found in the same chromosomal positions and with the same statistical significance indicating that different genes are associated with these traits.
Comparative genetic mapping in salmonid species has revealed homologous regions of the genomes of Atlantic salmon, rainbow trout, and Arctic charr (Danzmann et al.   (Phillips et al. 2009) and Arctic charr linkage group 18 (AC-18) (Danzmann et al. 2005), whereas Ssa10 corresponds to RT-8q and RT-27q (equivalent to chromosomes 5q and 2q, respectively) (Phillips et al. 2009) and Arctic charr linkage groups 4, 16, and 40 (AC-4, AC-16, and AC-40) (Danzmann et al. 2005). Genome-wide significant QTL for early male sexual maturation were mapped to RT-8, RT-17, and RT-24, whereas chromosome-wide QTL for this trait was found on RT-3 and RT-19 (Haidle et al. 2008). Martyniuk et al. (2003) did report an association (p=0.043) between microsatellite locus OmyRGT1TUF on RT-5 and precocious maturation and noted that one allele was associated with both a higher body mass and precociousness. However, this association was not considered significant after a sequential Bonferroni correction. Similarly, no p values less than 0.01 were identified for microsatellite marker alleles segregating with male sexual maturation in Arctic charr, although SSOSL32 on AC-4 gave a suggestive value of 0.014 (Moghadam et al. 2007). It is believed that sexual maturation in salmonids depends on the ability of the fish to reach certain developmental thresholds during a "critical period" over the winter/spring (Adams and Thorpe 1989;Thorpe 1994), in a direct relationship with somatic growth and/or energy storage during this period (Taranger et al. 1999). Further investigation of genotype-environment interactions are required to improve Atlantic salmon breeding programs. Photoperiod manipulation using artificial light exposure has proven effective as a means of preventing Atlantic salmon from reaching an anticipated sexual maturation (Endal et al. 2000;Oppedal et al. 2006), and this procedure is used with many other farmed fish (reviewed by Taranger et al. 2010). A clear link between feeding and age of sexual maturation has been described in salmonids (Shearer et al. 2006;Silverstein et al. 1998;Taranger et al. 2010;Thorpe et al. 1990), suggesting that by restricting rations, energy storage and, in particular, lipid stores decrease, thereby delaying sexual maturation (Duston and Saunders 1999;Rowe et al. 1991;Shearer et al. 2006;Shearer and Swanson 2000). This also explains the increasing use of tetradecylthioacetic acid to reduce early sexual maturation in Atlantic salmon (Arge et al. 2012) as it affects the β-oxidation of fatty acids, reducing levels of plasma lipids and adipose lipid stores, and enhancing transport of fatty acids to the liver.

Candidate Genes
To identify candidate genes associated with the traits of early or late sexual maturation, we made use of the currently available information from the Atlantic salmon genome sequencing project (Davidson et al. 2010), which is publicly available at ASalBase (www.asalbase.org). Most of the SNPs in the 6. 5K array correspond to an expressed sequence tag (EST) or are linked to a specific genomic sequence, and these sequences were assigned to a specific whole genome shotgun (WGS) contig by sequence similarity searches. WGS contigs were then annotated using an in-house annotation pipeline (trutta.mbb.sfu.ca) (see Table 3).
There are four SNPs linked to the QTL associated with grilsing on Ssa10, of which two could be linked to two genes by a BLASTn search (Altschul et al. 1990) and subsequent sequence annotation. These genes were a leukocyte elastase inhibitor (SERPIN-like) and a GDP-L-fucose synthase-like protein. We cannot speculate how these genes may be related to grilsing in Atlantic salmon. In the case of the SNPs linked to the grilsing QTL on Ssa21, one is associated with a gene encoding a formimidoyl transferase-cyclodeaminase, a protein related to metabolic processes. Two other SNPs (from the same EST) identified E3 ubiquitin-protein ligase (UBR3), a protein that has been shown to have a regulatory role in sensory pathways, including olfaction in mammals (Tasaki et al. 2007). The QTL detected for the late-maturation trait on Ssa18 was linked to two SNPs, but only one of them could be related to a known gene, namely, myopalladin-like gene. Studies in pig and cattle suggest  that this gene is associated with meat quality and carcass traits including fat content (Chong et al. 2007;Jiao et al. 2010).

Conclusions
In this study, we were able to identify a limited number of QTL associated with age at sexual maturation in Atlantic salmon. Moreover, our results suggest that these traits are controlled by genes independently from growth-related traits. Age at sexual maturation is a trait that has been heavily selected and improved after several generations of selective breeding based on family selection and individual selection (Gjedrem 2000;Gjerde 1984;Gjøen and Bentsen 1997). However, these improvements have been based on phenotypic observations, and thus, the identification of genetic markers and genomic regions associated with grilsing and late sexual maturation provides new resources for Atlantic salmon selective breeding programs.


extfieldsofstudy: Biology Medicine

text:
Luteinizing hormone and human chorionic gonadotropin: distinguishing unique physiologic roles

Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are integral components of the hypothalamic–pituitary–gonadal axis, which controls sexual maturation and functionality. In the absence of signaling through their shared receptor, fetal sexual differentiation and post-natal development cannot proceed normally. Although they share a high degree of homology, the physiologic roles of these hormones are unique, governed by differences in expression pattern, biopotency and regulation. Whereas LH is a key regulator of gonadal steroidogenesis and ovulation, hCG is predominantly active in pregnancy and fetal development. Emerging evidence has revealed endogenous functions not previously ascribed to hCG, including participation in ovulation and fertilization, implantation, placentation and other activities in support of successful pregnancy. Spontaneous and induced mutations in LH, hCG and their mutual receptor have contributed substantially to our understanding of reproductive development and function. The lack of naturally occurring, functionally significant mutations in the β-subunit of hCG reinforce its putative role in establishment of pregnancy. Rescue of reproductive abnormalities resulting from aberrant gonadotropin signaling is possible in certain clinical contexts, depending on the nature of the underlying defect. By understanding the physiologic roles of LH and hCG in normal and pathologic states, we may better harness their diagnostic, prognostic and therapeutic potential.

Introduction
Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) serve different roles in reproductive physiology: LH contributes to the driving force behind gonadal steroidogenesis and regulation of ovulation, whereas hCG (in the absence of pathologic abnormalities) is secreted in large amounts only during pregnancy [1]. The expression profile differences manifest early in fetal development and are maintained throughout the life span [2][3][4]. Although LH and hCG share the same receptor, many of their actions remain distinct [1,5,6]. A wealth of information regarding the endogenous functions of LH and hCG has been ascertained through naturally occurring and induced mutations, yet data are still emerging that shed new light on the involvement of LH and hCG in various physiologic processes.
By understanding how LH and hCG affect normal and abnormal human development and reproduction, we may be able to improve on current treatments for reproductive disorders while uncovering other areas where assessment of these gonadotropins may be of use, such as the management of certain cancers. This review describes the current state of knowledge regarding the physiology of LH and hCG throughout the stages of life and the impact of disrupting their normal expression profiles and functionality.

Fetal period
Fluctuations in circulating LH and hCG concentrations are observed throughout fetal development ( Figure 1) [1,3,6,7]. In the male fetus, initially high levels of plasma hCG decrease sharply between weeks 10 and 20 and then gradually decline for the remainder of gestation [6]. Expression of LH, in contrast, increases from week 10, reaching a peak before week 20 and decreasing slowly thereafter. The amount of hCG present at its nexus far exceeds that of LH [1,6]. Although fetal cells that are destined to become Leydig cells begin to produce testosterone as early as 6-7 weeks of gestation, it is not until after this period that hCG and LH are believed to be important to the process [8]. Indeed, the LH/ choriogonadotropin receptor (LHCGR) is not detected in Leydig cells until 10-12 weeks after conception [6]. As the more abundant and biologically active species, hCG likely plays a larger role in testosterone production by Leydig cells than does LH during early fetal development [6,9]. Testosterone is responsible for virilization of the reproductive tract, promoting the formation of the ductus deferens, epididymides, seminal vesicles and ejaculatory ducts [1,6]. Dihydrotestosterone, a derivative of testosterone, is active in the differentiation of the prostate and external genitalia. At 15-20 weeks of gestation, testosterone regulation shifts to the fetal pituitary gland, as LH secretion takes over from placental hCG in driving testosterone production [9]. This transition is evident in LH-deficient anencephalic male fetuses, in whom normal masculinization of the reproductive tract occurs while high levels of hCG are present, but further development of the external genitalia is hindered by the lack of LH when hCG levels decrease [9]. The pattern of pituitary LH expression during fetal development mirrors that of serum LH levels [3].
Peak concentrations of pituitary and circulating LH are higher in female fetuses relative to that of males [3,7]. It has been postulated that negative feedback from testosterone and inhibin in males lead to their lower LH levels, which is borne out by the higher levels of testosterone and inhibin B detected during gestation in males compared with females [7]. Development of the female fetal reproductive tract does not depend on the presence of LH or hCG. In fact, LHCGR is not expressed by the developing ovary until after the 16th week of gestation [6]. In general, minimal steroidogenesis occurs in the ovary during fetal development.
Detection of LHCGR in fetal organs, including the kidney, liver, lung, pancreas and gastrointestinal tract, has led to speculation that hCG may have a further role in fetal organ growth and differentiation [5,10]. No specific function in fetal organ development has yet been ascribed to hCG [5].

Infancy and childhood
In term infants, circulating LH and hCG concentrations are below the limits of detection [7]. After delivery, both sexes experience a surge in gonadotropin levels due to the withdrawal of maternalderived estrogen. The magnitude of LH elevation is greater in male than in female infants [2,11,12]. In male infants, LH peaks at 1-3 months of age, declining rapidly thereafter and reaching a nadir at 4-9 months of age. Testosterone levels, which are also elevated at 3 months of age in boys, decrease more precipitously, falling below the limit of detection by 6 months of age [12]. The testosterone peak may be driven by a coinciding increase in Leydig cell number and is believed to trigger germ cell proliferation and sexual differentiation of the central nervous system.
Infant girls demonstrate greater variability in LH expression patterns [12]. Generally, a peak in LH concentration is reached at 1-3 months of age, followed by a decrease to childhood levels and a possible small resurgence at 1 year [2,12]. The significance of transient hypothalamic-pituitary-gonadal axis activation is as yet unknown. After the temporary surge in boys and girls, gonadotropin levels remain low until prepuberty. This period is sometimes referred to as the juvenile pause [6,13].

Prepuberty and puberty
In the year or two preceding puberty, the gonadotropin-releasing hormone pulse generator begins to mature and escape its central inhibition. In response, there is a gradual increase in the frequency and amplitude of nocturnal LH pulsatile secretion [14,15]. With pubertal progression, this diurnal pattern of LH secretion abates, and LH is released in a more regular pulsatile manner throughout the day [13,16]. Concurrently, the proportion of bioactive to immunoreactive LH increases [17,18]. Phillips et al. [19] studied the shift in LH and follicle-stimulating hormone (FSH) isohormone profile in puberty. In boys, there was an increased prevalence of acidic FSH and LH isoforms observed at pubertal stage II. In contrast, the median charge of LH was largely unchanged in girls during pubertal stages I through IV.
The increase in gonadotropin activity during puberty drives gonadal steroidogenesis -primarily the production of testosterone by Leydig cells in males and testosterone and progesterone by thecal cells in females [1]. Most of the testosterones generated by thecal cells are converted to estradiol by granulosa cells. Testosterone, estradiol and adrenal androgens produce the physical changes associated with puberty, including the development of secondary sex characteristics. As estradiol production increases in girls, stimulation of the endometrium occurs, eventually leading to menarche (2-3 years after the onset of puberty). The hypothalamic-pituitary-ovarian axis matures in middleto-late puberty, with the development of estradiol's positive feedback effect on stimulating an LH surge [13]. More regular, ovulatory cycles do not become established until several years after menarche.

Adulthood
In women, LH and hCG play crucial roles in ovulation and pregnancy (discussed in the next section). After puberty, the pattern of LH secretion during the menstrual cycle becomes more regular in normo-ovulatory women. In women with polycystic ovarian syndrome (PCOS), however, higher basal levels of LH are observed with increased pulse frequency and amplitude throughout the cycle [20][21][22]. The lack of variation in LH secretion pattern contributes to the anovulatory cycles frequently found in women with PCOS.
In men, LH stimulates testosterone production by Leydig cells. Secretion of LH is pulsatile, but it does not demonstrate the cyclic variations observed in women. Low levels of hCG (presumably of pituitary origin) are also present in men [4], the function of which is unknown. Although not the key impetus for endogenous steroidogenesis, in conditions such as male hypogonadism and infertility, exogenous hCG may be administered to drive testosterone production [1].

Menopause and post-menopause
A decline in ovarian function during the perimenopausal period, which can last from 2-8 years [13], is marked by a gradual increase in FSH, a pronounced decrease in the follicular pool, and a reduction in early follicular phase inhibin secretion [1]. As menopause approaches, estradiol and estrone levels decline [1]. The subsequent lack of ovarian steroid negative feedback results in a sharp increase in LH and FSH secretion, with a gradual decline during the post-menopausal period [1,6].
During the perimenopausal period, serum hCG levels increase substantially in women [23]. The finding that hCG measurements in women 55 years of age and older are significantly greater than those in non-pregnant women aged 18-40 years or 41-55 years (p50.0001) could have diagnostic implications when interpreting hCG tests. After the onset of menopause, hCG levels seem to stabilize. The elevated levels of hCG in perimenopausal and menopausal women are believed to be pituitary in origin [23]. It should be noted that LH and hCG levels during and after the menopausal transition demonstrate a high degree of interindividual variability.
Although loss of reproductive capability akin to female menopause does not occur in men, they do experience a progressive decline in spermatogenesis and fertility with age. Leydig cells decrease in number and are less capable of producing testosterone in response to LH or hCG stimulation [6,24]. Aging is generally associated with a decrease in circulating total and free serum testosterone levels, but this phenomenon is not observed in all men [6,24,25]. On an average, testosterone levels decrease by 1-2% per year [25]. Levels of LH and hCG, in contrast, gradually increase with age [25,26]. The increases in LH and hCG levels in men are much less pronounced than those observed during the perimenopausal and post-menopausal intervals in women. Nonetheless, reference ranges for normal LH and hCG values in men are typically stratified by age group.

Roles of LH and hCG during ovulation and pregnancy
Ovulation Luteinizing hormone regulates ovulation through changes in expression levels and the proportions of glycosylated isoforms. Both LH and hCG exist as compilations of isohormones that differ with respect to post-translational modification (predominantly glycosylation) and state of degradation, which influences their biological activity and half-life in circulation [27,28]. Early in the follicular phase, estradiol exerts a negative feedback effect on LH; however, some LH activity occurs to stimulate thecal cell production of androgens that serve as substrates for estradiol production by granulosa cells. As estradiol levels rise in the late follicular phase, the relationship with LH becomes one with a positive feedback effect, leading to the LH surge and subsequent ovulation [6]. This transition corresponds with a shift toward less glycosylated and more biologically active LH isoforms [29,30]. With ovulation, the ovulatory oocyte progresses through to meiosis II while the corpus luteum forms in the ovarian stroma. LH drives progesterone production and secretion from the corpus luteum and, if pregnancy occurs, hCG takes over progesterone regulation. The hyperglycosylated isoform of hCG, which is abundant in early pregnancy, does not seem to play a role in the promotion of progesterone secretion [31].
Low levels of pituitary hCG parallel the dynamics of LH secretion during the menstrual cycle [4]. Pituitary hCG secretion is responsive to stimulation by GnRH analogs and is suppressed by oral contraceptives [32][33][34]. The role of this pituitary hCG is unclear, but the pattern of expression is consistent with overlapping function to that of LH [5]. It has been suggested that hCG expression may elevate the peak range of LH, thereby aiding in the promotion of ovulation [35]. In addition, lingering hCG could enhance progesterone production at luteal phase onset.

Fertilization and implantation
The presence of LHCGR in human fallopian tubes and in sperm suggests a role for LH and hCG in fertilization [36,37]. Consistent with this putative function, expression of LHCGR is greater in fallopian tubes during the luteal phase compared with the proliferative phase of the menstrual cycle or fallopian tubes from postpartum or post-menopausal women [36]. Studies using a porcine model demonstrated that LH was capable of inducing relaxation of the oviduct, most notably during the periovulatory interval [38]. It is not known what the function of gonadotropin signaling is in sperm; however, a recent study reported correlations between polymorphisms in LHCGR and sperm quality (e.g. concentration and motility) in cattle [39]. Investigators have speculated that hCG present in the female reproductive tract could activate the LHCGR on sperm, thus promoting motility and capacitation [37].
Blastocyst secretion of hCG may contribute to endometrial cross-talk to enable implantation [40,41]. Endometrial expression of LHCGR increases mid-luteal phase, during a short period in which the endometrium is receptive to implantation (i.e. the implantation window) [41]. Investigators hypothesize that a blastocyst capable of producing locally high levels of hCG could extend the implantation window, thereby increasing the chances for successful pregnancy [40]. A similar phenomenon of increased endometrial LHCGR expression during the implantation window has been observed in mice [42]. Given that the mouse genome does not include a chorionic gonadotropin gene, LH would need to functionally substitute for hCG in this system. Preliminary evidence suggests that this may indeed be the casea bioactive LH signal has been reported at the time of murine blastocyst implantation.
During early pregnancy, hCG is also believed to support implantation and placentation by modulating endometrial tissue remodeling, fostering maternal immunotolerance of fetal tissue, promoting neoangiogenesis and increasing the uterine natural killer cell population [5,40,41]. These functions all contribute to endometrial receptivity -a key component to successful pregnancy. Emerging evidence suggests that hCG expressed by the blastocyst stimulates receptors on the endometrium, eliciting a variety of effects on downstream molecules, including growth factors (vascular endothelial growth factor [VEGF], transforming growth factor beta), cytokines (leukemia inhibitory factor) and proteinases (matrix metalloproteinase 9 [MMP-9]), which contribute to different aspects of endometrial receptivity [40,41,43]. For example, hCG-mediated upregulation of VEGF is believed to promote angiogenesis, whereas stimulation of MMP-9 expression by hCG is involved in regulation of tissue remodeling. The role of hCG in fostering endometrial receptivity appears to begin even before embryonic hCG expression. It has been shown that epithelial hCG is expressed and produced in human endometrium biopsy specimens during the early to mid-secretory phase of the menstrual cycle [44]. Elucidating the precise role of hCG in endometrial functions related to implantation remains an active subject of investigation.

Maintenance of pregnancy
Expression of hCG continues throughout pregnancy, with peak levels occurring at 10 weeks' gestation [5]. As many as 20-30 isoforms of hCG can be detected during pregnancy [45]. Hyperglycosylated hCG (hCG-H) secreted by root or extravillous cytotrophoblasts is the predominant species early in gestation, but it is gradually surpassed in proportion by regular hCG, which is produced by fused and differentiated syncytiotrophoblast cells [5]. It has been well established that in early pregnancy, hCG sustains progesterone production by the corpus luteum. Placental production of progesterone takes over in the later part of the first trimester, at a time when hCG levels are in decline [6]. Beyond its role in luteal support, hCG is believed to promote angiogenesis, thereby supporting blood flow to the placenta and nutrition for the fetus [46,47]. Emerging evidence also suggests that hCG is involved in umbilical cord growth and development, synchronizing uterine growth with that of the developing fetus and relaxation of myometrial contractions [5]. In addition, the presence of LHCGR in the brain has led to speculation that gonadotropin signaling is involved in the nausea and vomiting experienced during pregnancy [48].
Quantification of hCG species is a valuable diagnostic and prognostic tool during pregnancy. For example, measuring hCG-H levels, which increase rapidly during the initial stages of pregnancy, allows for earlier detection than standard pregnancy tests. A recent study showed that an ultrasensitive hCG-H assay was capable of detecting pregnancy 6 days after embryo transfer in women undergoing in vitro fertilization [49]. At 12 days, lower levels of hCG-H distinguished biochemical from clinical pregnancies. Insufficient hCG-H levels (i.e. urinary hCG-H550% of total hCG) have also been detected in a large proportion of early pregnancy losses in spontaneously conceived cycles [50]. Low circulating concentrations of another form of hCG, the disassociated b-subunit, have been linked to an increased probability of ectopic pregnancy in symptomatic patients [51]. During the later stages of pregnancy, inadequate amounts hCG-H are associated with the development of preeclampsia [52]. Preeclampsia itself is attributed to abnormal placentation and trophoblast invasion [53]. On the other end of the spectrum, elevated levels of hCG-H or hCG free b-subunit in maternal serum during the first trimester are markers of Down syndrome pregnancy [54]. Triploid abnormalities may be discernible as early as the pronuclear stage by the presence of hCG-H [55]. Notably, most methods currently used for hCG quantification in clinical laboratories underestimate hCG-H levels.

Pathology of LH, hCG and LHCGR
Loss-or gain-of-function mutations have helped to establish the extent to which gonadotropin signaling is necessary for sexual development and reproductive success, while also providing insights into pathologic processes. These gene alterations may be naturally occurring, as is the case for human mutations/polymorphisms, or induced in model systems (most commonly in mice). The effects of targeted mutations in LH or overexpression of hCG on reproductive structure and function in mice are presented in Table 1. A description of human phenotypes associated with changes in LH, hCG and LHCGR functionality or expression are provided below and summarized in Table 2.

Human mutations/variants
LHB Naturally occurring mutations that result in inactive LH are rare in humans, particularly in women. Only two cases in female patients with inactivating LHB mutations have been described to date [63,64]. Characterization of one of these individuals revealed normal development and appropriate pubertal milestones followed by secondary amenorrhea and infertility [63]. Uterine volume was normal, although the endometrium was atrophic. The absence of detectable LH did not seem to adversely affect FSH, hCG, testosterone, or prolactin levels; estradiol and progesterone levels were in the low-to-normal range for the follicular phase of the menstrual cycle. Supplementation with estrogen and progesterone was capable of inducing endometrial thickening and follicle enlargement; however, normal ovulation was not restored and no corpus luteum was observed. The second patient was reported to have experienced menarche at age 14 years, with subsequent oligomenorrhea and secondary amenorrhea [64]. Residual LH activity described in an affected male sibling suggests that this mutation did not result in the complete absence of LH, although serum levels were below the limit of detection. The reproductive findings in these women confirm that adequate DOI: 10.3109/09513590.2013.859670 LH is not required for normal sexual differentiation and pubertal development, but it is necessary for ovulation and development of a corpus luteum.
Men with homozygous inactivating LHB mutations have a more severe phenotype characterized by hypogonadism, severe testosterone deficiency and azoospermia [63,65,66]. Leydig cells are absent or hypoplastic. Sexual differentiation and development proceed normally through gestation and childhood, but pubertal maturation does not occur. Male heterozygotes display a milder phenotype, with normal pubertal development but altered steroidogenesis and increased risk of infertility [65]. Long-term treatment with hCG is capable of rescuing the hypogonadal phenotype in men with inactivating LHB mutations, resulting in virilization, increased testicular volume, elevated testosterone levels and improved spermatogenesis [65][66][67]. Restoration of spermatogenesis allowed one of these men to father a child through intracytoplasmic sperm injection [67]. There has been one reported case of an LHB mutation that does not completely abrogate LH activity and is compatible with complete spermatogenesis even in the absence of virilization and in the presence of low serum testosterone levels [64]. Despite a reduced number of Leydig cells compared with an age-matched control, sperm production in this patient was quantitatively normal. These observations suggest that the LH threshold for sperm production is relatively low.
One common variant of LHB (often referred to as ''variant LH'') has been detected in 7.1-41.9% of the world's population [82,83]. This polymorphic isoform contains two point mutations, resulting in a functional LH with a shorter half-life and possibly decreased bioactivity relative to normal LH. Amino acid sequencing of variant LH suggests a closer homology to hCG than to normal LH. Current LH antibody assays are less able to Prepubertal external genitalia, reduced testes size, impaired spermatogenesis, testosterone deficiency, infertility LH receptor null [57,58] Female Underdeveloped genitalia, ambiguous vaginal opening, arrested follicular growth, decreased estradiol and progesterone levels, infertility Male Underdeveloped genitalia, abdominal testes, micropenis, Leydig cell hypotrophy, disarray of seminiferous tubules, spermatogenic arrest, modestly elevated estradiol levels, infertility LH b-subunit null [59] Female Hypogonadism, decreased serum estradiol and progesterone levels, defects in folliculogenesis, gene expression abnormalities, uterine hypoplasia, infertility Male Decreased testicular size, Leydig cell hypoplasia, gene expression abnormalities, reduced testosterone levels, impaired spermatogenesis, infertility hCG a-subunit overexpression [60] Female and male Normal and fertile* hCG b-subunit overexpression [60][61][62] Female Precocious puberty, enhanced ovarian steroidogenesis, abnormal uterine structure, hyperprolactinemia, infertilityy Male Mild hypogonadism, fertiley Infertility* hCG aand b-subunit overexpression [60,61] Female Elevated levels of serum estradiol, hemorrhagic and cystic ovaries, thecal layer enlargement, stromal cell proliferation, infertility*, tumorigenesis* Male Increased testicular androgen production, focal Leydig cell hypertrophy, progressive seminiferous tubule degeneration, urethral obstruction, infertilityy Leydig cell hyperplasia, very high serum testosterone levels, reduced testis size, enlarged seminal vesicles, infertility* LH, luteinizing hormone; hCG, human chorionic gonadotropin. *Transgene expression was driven by the ubiquitin promoter. yMice expressing high levels of hCG, with transgene expression driven by the metallothionein 1 promoter. detect variant LH, which may pose difficulty in ovulation testing. In certain patient cohorts, variant LH has been linked to ovulatory disorders, including hyperprolactinemia, premature ovarian failure, menstrual disorders, luteal insufficiency and PCOS [68][69][70]. Variant LH allele carriers may be at increased or reduced risk for developing ovulatory dysfunction, depending on the specific disorder and population evaluated. Separate (non-variant LH) polymorphisms have also been associated with PCOS susceptibility and phenotype [71,72], as well as with endometriosis, premature ovarian failure and luteal insufficiency [71].
hCG Sequence changes that influence the function of hCG are rare. It has been suggested that mutations with a significant effect on hCG would not be compatible with successful pregnancy and are, therefore, not observed in the population at large [73]. This hypothesis is consistent with the postulated importance of hCG signaling from the blastocyst for implantation and the additional functions of hCG during gestation. It seems intuitive then that polymorphisms in the hCG b-subunit (i.e. the CGB gene) are associated with increased risk of recurrent miscarriage [73].

LHCGR
A variety of naturally occurring activating and inactivating mutations have been identified in the LHCGR gene. In men, activating mutations of LHCGR result in autosomal dominant familial male-limited gonadotropin-independent precocious puberty (i.e. testotoxicosis) [74]. Affected individuals develop Leydig cell hyperplasia and demonstrate evidence of pubertal development in early childhood. Of the variety of gain-of-function LHCGR mutations reported, only D578H is known to cause Leydig cell adenomas in addition to precocious puberty [84]. Whereas the other LHCGR activating mutations have a familial or germline origin, D578H is somatic [74]. Surprisingly, women with activating LHCGR mutations display no reproductive abnormalities in structure or function. Women with inactivating mutations of LHCGR, on the other hand, have a similar phenotype to that of inactivating LHB mutations, including oligomenorrhea/ amenorrhea and infertility [75]. In addition, an LHCGR mutation that is believed to reduce receptor expression and binding capacity has been implicated in empty follicle syndrome [76]. Bentov et al. [85] recently reported a reproductive phenotype in a woman who was heterozygous for an inactivating LHCGR mutation. Previously identified loss-of-function LHCGR mutations had only generated overt reproductive pathology in the homozygous state (i.e. autosomal recessive inheritance). This patient presented with amenorrhea and had a history of irregular periods, endometriosis and recurrent ovarian cysts. Attempts at in vitro fertilization resulted in poor egg yield and multiple biochemical pregnancies.
Inactivating mutations of LHCGR cause Leydig cell hypoplasia in men [74]. There are degrees of retained LHCGR activity among the inactivating mutations, which influences the patient's phenotype. Genetically, males with complete loss of LHCGRmediated signaling develop Type I Leydig cell hypoplasia, wherein male sexual differentiation does not progress normally during fetal development, resulting in psuedohermaphroditism. Partial loss of LHCGR function yields a milder and more variable form of Leydig cell hypoplasia (Type II) that may include micropenis and hypospadias. In one unusual case of Leydig cell hypoplasia Type II, a patient presented with hypogonadism, azoospermia and pubertal delay, despite otherwise normal prepubertal male development [86]. Functional analysis revealed that the LHCGR mutation in this patient rendered the receptor insensitive to LH but not to hCG [87]. Consequently, unlike typical inactivating LHCGR mutations, treatment with hCG was effective for treating hypogonadism and restoring spermatogenesis in this patient [86]. This case underscores the inherit divergence in the physiologic roles of endogenous hCG and LH: continued hCG sensitivity allowed for normal fetal sexual differentiation, but lack of LH response prevented pubertal development and sperm production.
Sequence alterations in components of the gonadotropin signaling pathway have also been linked to cancer risk and prognosis. Polymorphisms in LHCGR, for example, are associated with an increased risk of testicular germ cell cancer and endometriod adenocarcinoma [77,88], whereas variant LH is considered a weak risk factor for prostate cancer [78]. Moreover, increased LHCGR activity resulting from a common polymorphism has been correlated with shortened disease-free survival in patients with breast cancer [79,80]. Investigators postulate that the negative influence on cancer outcome of this LHCGR allele results from an increase in estrogen exposure. Further investigation is needed to better understand the role of aberrant gonadotropin signaling in cancer etiology.

Conclusions
Despite a high level of homology and action at a common receptor, LH and hCG play unique roles in development and reproduction. LH and hCG act during specific windows of opportunity during fetal and post-natal development, at times coordinately, to promote proper sexual differentiation, maturation and functionality. Although long known to support pregnancy through its effect on progesterone production, hCG is increasingly recognized for its multitude of activities that foster implantation and subsequent maintenance of pregnancy. Loss-of-function mutations prove that the absence of one gonadotropin is not fully compensated by endogenous production of the other, yet rescue through exogenous administration is possible. Success of supplementation with hCG or downstream hormones in the gonadotropin cascade for spurring pubertal development, treating hypogonadism or managing infertility depends on the nature of the underlying molecular abnormality. The ongoing study of LH and hCG physiology will aid in the development of diagnostic tests, prognostic assessments and therapeutic approaches relevant to human disease and reproduction.

Declaration of interest
Financial support for manuscript development was provided by Ferring Pharmaceuticals, Inc., Parsippany, NJ. Crystal Murcia, PhD, and Jill McCollam, PharmD, of The JB Ashtin Group, Inc., prepared this manuscript for publication based on the authors' initial draft and direction. The authors alone are responsible for the content of this article. The authors report no conflicts of interest.