Limodromus assimilis. The genital tract consists of pairs of uni-follicular testes, spermatic ducts with diverticula regions, seminal vesicles, accessory glands, an unpaired ejaculatory duct and an aedeagus containing an internal sac equipped with sclerotic scales. Based on their morphology, we draw conclusions about their functions. After spermatogenesis within the follicle, the spermatozoa become released from the sperm cysts. The single spermatozoa move into the diverticula of the vasa deferentia I. Here, they become attached to central rods (spermatostyles), forming secondary conjugates (spermiozeugmata). The coordinated flagella movement of the conjugates possibly improves sperm velocity. Using super-resolution microscopy, we identified highly condensed reticulate chromatin in the lancet-shaped spermatozoa heads and the mitochondrial derivates of the flagella, likely formed by genomic and mitochondrial DNA, respectively. The results show, for the first time, sperm bundle formation in a Platynini species mainly corresponding to that found in Pterostichini species.Based on advanced light and electron microscopy, we describe the male reproductive system and sperm development of  Gilson [Insect reproductive systems show large morphological variability. Similarly, their spermatozoa may vary strongly in shape and size , 2. In aGilson . These wGilson , HymenopGilson , OrthoptGilson  and ColeGilson .Within the Carabidae, spermatozoa morphology has already been analysed in the taxa Cicindelinae , ScaritiPterostichini, the flagella are completely movable within the conjugate, or they are attached to the spermatostyle [Abax parallelpipidus Piller et Mitterpacher 1783 (Carabidae: Pterostichini), producing a 17-times twisted spiral formed by a central spermatostyle where spermatozoa are attached [Carabus insulicola Chaudoir 1869 (Carabinae), endemic to Japan, forms heteromorphic sperm conjugates in which the spermatostyles, where spermatozoa associate, are of varying lengths [Jujiroa estriata Sasakawa, spermiozeugmata were analysed. This species produces 2.4-mm long bundles containing movable flagella [Within the atostyle . In someatostyle , and theattached . Short,  lengths , 12. UntThe reason some species form sperm bundles is still under debate. They seem to be essential for increasing the sperm transfer efficiency to the female during copulation. It is thought that spermatozoa aggregation improves their mobility compared to that of single spermatozoa , 21. In A. parallelpipidus and Pterostichus nigrita (Paykull 1970), indicate that in these structures, the formation of spermiozeugmata appears [Higginson and Pitnick [Each of the twin testis of Adephaga (Coleoptera), thus also of Carabidae, is formed by only one raised follicle  containi appears , 19, 26. Pitnick  describeIn some conjugate-forming Carabidae species, the mature spermatozoa are released from the sperm cysts, are transferred via the short vas efferens into the diverticulum of the vas deferens region, and are attached here to the spermatostyle. The spermatostyle becomes secreted from the diverticulum epithelium, and after reaching a defined length, it becomes released from the diverticula. Then, spermiozeugmata are transported to the seminal vesicle to be stored until copulation , 19, 26.Colymbetes fuscus (Linnaeus 1758) (Coleoptera: Dytiscidae) are connected via proteins and polysaccharides [Parachauliodes japonicus (McLachlan 1867)  and P. nigrita (Carabidae) [The chemical composition of Carabidae spermatostyles is not yet clear. The double spermatozoa of charides . Proteinrabidae) , 29.Will et al. [Accessory glands, also belonging to the male reproductive system, may be present in different numbers and shapes in Coleoptera species . In Harpl et al.  found twl et al. . The mail et al. . In mostl et al. .Limodromus assimilis (Paykull 1790), which belongs to the Platynini tribe also containing sperm bundles [Previously, the morphology and formation of Carabidae spermiozeugmata were mainly described within the tribe Pterostichini originating from East Asia . It was  bundles . Due to  bundles . The proL. assimilis. Based on these observations and simple staining procedures delivering first rough hints on the chemical components of the reproductive organs and tissues, we conclude their functions.In the present work, we describe the morphology of the male reproductive system, spermiozeugmata formation and the spermatogenesis of L. assimilis individuals  under long-day conditions  at 23°C in order to positively influence the maturation of the genital organs. Romeis [The beetles were killed with chloroform, and the dissected organs were put into Ringer’s solution to remove the trachea, Malpighian tubules and fat bodies. Spermiozeugmata were obtained by scratching out the seminal vesicles in Ringer’s solution. The genital organs were fixed in Carnoy’s solution, treated in an ascending ethanol series  and isopropanol, and imbued with paraffin at 65°C. Using a Leica (SM 2010 R) microtome, the sample blocks were cut in 5- or 8-μm thin sections and then placed onto protein-glycerine treated slides. The deparaffinised samples were stained with haematoxylin and eosin (HE) after Romeis .Spicer and Meyer [The aldehyde fuchsin-alcian blue (AF-AB) staining was applied after nd Meyer . For theThe genital organ sections and the DAPI-stained spermiozeugmata were analysed under a Leica microscope (Leitz DMRBE) applying Differential Interference Contrast (DIC) and fluorescence microscopy, respectively. To analyse the structure of chromatin in spermatozoa heads and the mitochondrial derivates above the diffraction limit of light (super-resolution), spatial Structured Illumination Microscopy (3D-SIM) was applied using a C-Apo 63×/1.2W Korr objective of an Elyra PS.1 microscope system and the software ZEN (Carl Zeiss GmbH) to achieve a lateral resolution of ~120 nm and an axial resolution of ~250 nm. Images were captured using a 405-nm laser for excitation and the appropriate emission filter to identify DAPI . SIM imaThe evaginated aedeagus was fixed in Carnoy’s solution, dehydrated with ethanol  and isopropanol and then fixed in hexamethyldisilazane according to Bock . The dryL. assimilis and Snodgrass [Kaulenas [Snodgrass [First, the follicle leads into a short vas efferens (defined by nodgrass ) followeKaulenas  and Snodnodgrass ), repres1). A thin epithelial cell layer surrounds the sperm cyst. The continuous spermatogonia generation induces their proximal movement. After mitosis completion, the spermatogonia nearly double in size and then perform meiosis as indicated by different stages . The resulting globular spermatids elongate during spermatogenesis starting with flagella formation, in which for now the chromatin (shown in violet by HE staining) remains in the globular sperm head . Afterwards, the chromatin becomes elongated, indicating the mitochondrial derivate formation along the flagella. The spermatid heads are disposed to the cyst membrane, where they seem to be attached . Later, in the head, the nucleus containing condensed chromatin becomes visible . The highly condensed genomic chromatin was identified in the spearhead-like shaped sperm heads. In addition, chromatin-free regions are evident in the sperm head nucleus  region of the vasa deferentia appears as an ~20-mm long spiral containing more than 400 diverticula. All these wall protrusions are outside of the spiral and their tips are oriented towards the vas efferens . The antThe spermiozeugmata comprise a central rod where up to ~200 spermatozoa are attached. Their mean length is ~335 μm . The sliThe second vas deferens region (II) does not contain diverticula, has a diameter of ~70 μm and is only slightly contorted. HE staining indicated that it consists of a single-layer columnar epithelium surrounded by a thin muscle layer . SimilarThe seminal vesicle is an ~4–6 mm long part of the vasa deferentia and has, depending on its filling state, a variable diameter of ~150–250 μm. Similar to vas deferens II, the wall consists of a single-layer columnar epithelium surrounded by a thin muscular layer. The seminal vesicle accumulates and stores a high amount of spermiozeugmata . After AFrom the seminal vesicle, the sperm bundles are transported to the accessory glands via a tight corridor which has a strong circular muscular layer. The accessory glands  are filled with a white viscous secretion. The inner wall epithelium (~12 μm thick) contains secretory adenocytes. This columnar gland wall epithelium is surrounded by circular and longitudinal muscle layers ~30 μm thick . An AB-PSperm bundles present in the accessory glands are transported via contraction of their double-layered musculature  into theDuring copulation, this sac becomes evaginated, and the ejaculate is released through the gonopore. The internal sac surface has sclerotic scales which are oriented basally. They are flat at the basal region and become spicule-like towards the gonopore . Scale sL. assimilis testes contain, in addition, a part of the vas deferens. As by all other insects [P. nigrita [A. parallelpipidus [Adephaga (Coleoptera) testes consist of a single tube-like coiled follicle (Lawrence & Britton 1994). Similar to the globular testes of two Sardinian Carabids (Pterostichini) ,  insects , the com nigrita  and A. pL. assimilis contains diverticula. However, they vary significantly in size and number. L. assimilis has four times more (~400) diverticula than Calathus fuscipes Goeze 1777 (Carabidae: Pterostichini), but they are ca. twice (~160 μm) as long [A. parallelpipidus, but these diverticula appear croissant-like [P. nigrita [L. assimilis, and their ovoid shape is similar to the globular protrusions of Percus strictus strictus Dejean 1828 [Similar as the spermatic duct of other Carabidae  the vas  as long . Similarant-like . Similar nigrita , divertiean 1828 .A. parallelpipidus [P. strictus strictus [Speomolops sardous Patrizi 1955 [P. nigrita [Calathus species [L. assimilis is responsible for sperm bundle formation. Each diverticulum contains one central rod and spermatozoa. We suggest that spermatozoa move from the anterior tract into the diverticulum where they attach at the central rod via their heads. Ferenz [P. nigrita. In addition, it could be that the diverticula contractions absorb the spermatozoa of L. assimilis.Probably, similar as that described for other Pterostichini, lpipidus , P. stristrictus , Speomolizi 1955 , P. nigr nigrita , and Cal species , the div. Ferenz  suggestsP. nigrita.The origin of the substances forming the spermatostyle is not yet clear. Indicated by the HE staining, they are possibly secreted from the diverticulum epithelia cells. The secretory function of diverticulum epithelial cells to produce central rod substances has been described for some Carabidae species , 19, 26.L. assimilis is not attached by sperm heads, probably because it is not accessible due to its embedding in the apical diverticulum epithelium when spermatozoa attach. Similarly, the spermiozeugmata heads of P. nigrita are fixed in a microvilli-rich impression of the epithelium in the diverticulum apex [The sperm bundle apex of lum apex .L. assimilis contains secondary sperm bundles, which should be called spermiozeugmata according to Higginson and Pitnick [Because sperm bundle formation proceeds after spermatogenesis is completed,  Pitnick . These sL. assimilis, we confirm the spermiozeugmata variability present in the family Carabidae, and for the first time, also within the tribe Platynini. J. estriata, belonging to the same tribe, shows a similar sperm bundle structure as L. assimilis, but its spermatostyle is filamentary and 15-times (2.4 mm) longer [Analysing ) longer .L. assimilis, species of the related tribe Pterostichini show mostly longer and convoluted sperm bundles [P. strictus strictus; [P. nigrita; [L. assimilis, the spermatozoa of many Pterostichini species are not arranged around the spermatostyle. Instead, they are attached as two strands at both sides of the central rod  or adhenigrita; ). In conral rod , [Pasimachus subsulcatus Say 1823 (Carabidae: Scaritinae) [Dendroctonus armandi Tsai and Li (Coleoptera: Scolytinae), these derivatives acquire different sizes and become laterally located in relation to the nucleus [Tribolium castaneum  (Coleoptera: Tenebrionidae) also two asymmetric mitochondrial derivatives were observed [Rhynchophorus ferrugineus Oliv. (Coleoptera: Dryophthoridae) contain mitochondrial derivates of different size, in which the large derivative is embedded into an infolding of the nucleus [L. assimilis the DNA-specific DAPI staining identified two identical parallel, tightly connected, and recessed mitochondrial derivates, which were found only along the posterior flagellum region. They show a finer reticulated structure, likely representing cristae, than the highly condensed head nucleus chromatin. Similar shaped mitochondrial derivates have not yet been found in other insects. Nevertheless, in despite of the different shape we suggest that they are required for sperm motility as has been found in Drosophila melanogaster Meigen, 1830 (Diptera) [At the end of meiosis, each spermatozoon receives an equal set of mitochondria. They aggregate and form the “nebenkern”, from which two mitochondrial derivates originate . In seveelinae),  and Pasiritinae) , two par nucleus . Similarobserved . Mature  nucleus . In contDiptera) .L. assimilis belong to the simplest type known among Coleopterans [Leptinotarsa decemlineata Say 1824 (Coleoptera: Chrysomelidae), Popillia japonica Newman 1841 (Coleoptera: Scarabaeidae) and in Cleridae and Carabidae species [L. assimilis show a curved shape, confirming the finding of Will et al. [The accessory glands (a pair of tubes) of opterans . This ty species , 47–49. l et al. . The glal et al. .L. assimilis. Contrary to L. decemlineata and P. nigrita, which exclusively include proteins and neutral mucines [L. assimilis additionally contains acid (carboxylated) mucosubstances.The secretion excreted by the epithelium of many insects mainly contains polysaccharides, proteins and mucosaccharides . The AB- mucines , 50, theL. assimilis individuals after copulation [L. assimilis, the gland lumen secretion is responsible for the formation of spermatophores, similar as is described for T. molitor (Coleoptera: Tenebrionidae) [L. assimilis glands they were identified only separately. The spermatophore formation could proceed in the posterior (glandular) parts of the vas deferens, similar as has been found by Krüger et al. [P. nigrita.The main accessory gland function of many insects is the formation of spermatophores , 51. Thepulation . Possiblionidae) . Howeverr et al.  in P. niL. assimilis, present also in P. nigrita [S. sardous [Snodgrass [L. assimilis as is present in all other insects [L. assimilis ejaculate contains complete spermiozeugmata because they are still present in the ejaculatory duct, and according to Fritzsch [The circular and longitudinal gland muscle layers of  nigrita  and S. s sardous , allow vnodgrass  the regi insects . It seemFritzsch , they haL. assimilis is required to transfer the ejaculate into the female during copulation [Sharp and Muir [L. assimilis resembles that of Carabus violaceus Linnaeus 1758 (Carabidae: Carabinae) [Similar to other insects, the aedeagus of pulation . Althougand Muir  for Cararabinae) .L. assimilis has sclerotic scales, but no additional spikes as is found in P. nigrita [L. assimilis seem to serve as brace organs during copulation. Due to an increased pressure caused by haemolymph during the in vivo copulation [The internal sac of many insects shows scales and spikes . That of nigrita , which a nigrita , the parpulation , the intS1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S1 Movie(MP4)Click here for additional data file.S2 Movie(MP4)Click here for additional data file.S3 Movie(MP4)Click here for additional data file.S4 Movie(MP4)Click here for additional data file.S5 Movie(MP4)Click here for additional data file.S6 MovieThe real time recording reflects a duration of 28 seconds.(MP4)Click here for additional data file.


Bulbophyllum weberi and B. cumingii are characterized by fly-pollinated features. The secretory activity was described in dorsal sepals in both species (putative osmophores), petals in B. weberi (possible osmophores) and adaxial surface of lips in both species. In the cells of dorsal sepals and petals of B. weberi proteins, dihydroxyphenols, lipids and starch grains were detected, in lateral sepals—lipids. Whereas in dorsal sepal of B. cumingii only lipids and starch grains were noted, in lateral sepals—proteins and dihydroxyphenols and in petals—proteins and starch grains. The lips in both species differed histochemically and ultrastructurally. The epidermal cells of lip groove in B. weberi contained lipids, proteins, starch grains in cytoplasm, dihydroxyphenols in vacuoles and pectic acids/mucilage on surface. Whereas in B. cumingii—few lipids, starch grains, no proteins, no dihydroxyphenols and no mucilage were noted. Ultrastructurally, in B. weberi, the secretory material was present on surface and vesicles building into plasmalemma, while in B. cumingii—cell wall ingrowths and microchannels in cuticle. The osmiophilic irregular materials and globular, osmiophilic globules in B. weberi are probably tannin-like materials. For the first time, we described the cell wall ingrowths in Bulbophyllum species: in lip of B. cumingii and petals of B. weberi.Flowers of  Bulbophyllum Lindl. is a large, pantropical genus, containing about 2200 species, distributed from Continental Africa, Madagascar, East Indian Islands, Asia, Australia and the tropical Pacific islands to the Neotropics. Madagascar and New Guinea are the main centres of diversity , but also by bees, wasps and foraging beetles  and ketones have been identified as key chemical components in plant-pollinator interaction. In B. cheiri methyl eugenol acts as sex pheromone precursor for males of Bactrocera species during pollination  and musky, slightly sweet or fruity odour. It is also known that myophilous flowers could produce secretion in open shallow nectaries which occur in longitudinal labellar groove in  Endress . Whereas Endress . In fruiBulbophyllum species has homogenous and highly conservative characters  Rchb.f.  Spreng., B. morphologorum Kraenzl., B. orientale Seidenf., B. wangkaense Seidenf. (Davies and Stpiczyńska B. wendlandianum (Kraenzl.) Dammer  Rchb.f., B. lupulinum Lindl., Bulbophyllum maximum (Lindl.) Rchb.f., B. schinzianum Kraenzl.  Benth. and Hook.f.), B. macranthum Lindl. (section Stenochilus J.J.Sm.), B. alticola Schltr. (section Codonosiphon Schltr.), B. auratum (Lindl.) Rchb.f. (section Recurvae). Pohl . Pohl  recordedJongejan . FlowersJongejan .Bulbophyllum species contains adaxial secretory tissue, which is defined in the literature as ‘nectary’. Teixeira et al. (Bulbophyllum. The liquid-like nectar was present in the lip cavity (=groove) of the non-wind-assisted fly-pollinated Bulbophyllum species: B. epiphytum (Barb. Rodr.) Cogn., B. glutinosum (Barb. Rodr.) Cogn., B. regnellii Rchb.f. and B. rothschildianum (O’Brien) J.J. Sm., whereas the osmophores were located as papillae on the lip lobes and in the upper surface of the lip callus in most investigated wind and non-wind-assisted species  J.J. Sm.: the movable lip with a trimethylamine-like odour and the upper groove with a viscous nectar. The fragrances generally are not accumulated on the surface  Dammer  as well as to discuss the results. The reasons to compare the results of both species are as follows: (1) B. weberi and B. cumingii are plants from the same section growing in the same range, (2) the same colours of flowers: yellow and purple, but in different arrangement, (3) coverage by papillae of dorsal sepals and petals, with some differences and (4) morphological similarities in lips shapes and features. The above-mentioned macromorphological features let us ask the question whether they are the same on anatomical level.The representatives from section B. weberi in November 2011 . Fresh flowers were observed under a Nikon SMZ1500 stereomicroscope (light microscope (LM)). Plant material was fixed in 2.5 % glutaraldehyde (GA) in 0.05 M cacodylate buffer (pH = 7.0). The material for LM was rinsed with cacodylate buffer and then dehydrated in acetone (for TEM) or ethanol series (for LM). Whole dehydrated material was embedded in epoxy resin (Spurr w/v) aqueous ruthenium red solution and a 10 % (w/v) aqueous solution of FeCl3 were used to test for pectic acids/mucilage , after dehydration in an ethanol series, the samples were dried by the critical point method using liquid COFor transmission electron microscopy (TEM), the floral material was fixed in glutaraldehyde (2.5 % GA) in 0.05 M cacodylate buffer (pH 7.0). The material was post-fixed overnight in 1 % OsO4 in cacodylate buffer in a refrigerator and then rinsed in the buffer. After 1 h in 1 % solution of uranyl acetate in distilled water, the material was dehydrated with acetone and embedded in Spurr’s resin. Ultrathin sections were cut on a Sorvall MT 2B ultramicrotome with a diamond knife and contrast stained with uranyl acetate and lead citrate. The sections were examined in a Philips CM 100 transmission electron microscope. Samples were prepared in accordance with procedures described elsewhere  and several vascular bundles  surface was consisted of longer conical papillae  and free ribosomes . Testing with SBB, lipoid layer on cells (cuticle) and few lipid droplets in cytoplasm were recorded . Some globules were stained on proteins . The TEM results of B. weberi showed that the striate cuticle was reticulate (with microchannels)  were stained on proteins  were those ones which stained positively on dihydroxyphenols  had plastoglobuli, electron-dense body and irregular profiles  Fig. h and coleth Fig. a. In B. pex Fig. a. The trB. weberi and B. cumingii are characterized by fly-pollinated features, i.e. colour, motile hair(s) at the tepals, motile and hinged lip. The overview of anatomical results upon both species revealed the secretory activity in the appendages and apices of dorsal sepals in both species (putative osmophores), petals in B. weberi (putative osmophores) and adaxial surface of lips in both species . The petals of B. cumingii are rather inactive in secretion process. The lipid layer on the apices of lateral sepals of B. cumingii may protect the tissue.Flowers of B. weberi were papillate and could function as osmophores. The term ‘unguentarius’—the floral organ bearing osmophores could be adopted here, as described in Utricularia dunlopii  and slightly on phenolic compounds and pectic acids, they were interpreted as putative osmophores. The floral sunken trichomes have also been noted on tepals and labellum in Maxillaria dichroma Rolfe, and described as resin-secreting trichomes  and this suggests that they have lipoid character. They could also be tannin-like materials  Rchb.f. and B. maximum Kraenzl.  (van der Pijl and Dodson Bulbophyllum species (defined as ‘nectary’) generally comprises a secretory epidermal cell layer and few subepidermal layers  Rchb.f. and on outer cell walls of B. schinzianum Kraenzl. , they appeared at the beginning of anthesis stage as small proturberances of the nectariferous epidermis and parenchyma formed from tangential walls of petals, and at full anthesis developed into labyrinthine protuberances. Such wall ingrowths, with the presence of microchannels or pores in cuticle, improve the nectar resorption. Resorbed nectar can be transported along the symplast and/or apoplast, and then is translocated across vascular tissue to the reservoir of assimilates , whereas cells in petal of B. weberi rather function as osmophore. The cell wall ingrowths described in Epipactis atropurpurea—species with hexose rich nectar, whereas in Limodorum abortivum were absent—species with sucrose dominant in nectar . The tannin-like material (dihydroxyphenols) found in cells of studied tepals offers protection against pathogens, herbivores and UV radiation. Tannins are described in cell suspension cultures and calluses from various gymnosperms (Constabel B. weberi. Localized under the epidermal cells, they probably protect from herbivory (Prychid and Rudall B. weberi demonstrated that epidermal cells contain lipids, proteins, starch grains in cytoplasm, dihydroxyphenols in vacuoles and pectic acids/mucilage on surface. Whereas, in B. cumingii—few lipids, starch grains, no proteins, no dihydroxyphenols and no mucilage were noted. Ultrastructurally, we found in B. weberi secretory material on surface, while in B. cumingii—cell wall ingrowths and microchannels in cuticle layer.The lip groove of on Fahn,  or otheron Fahn, . In B. cB. weberi and B. cumingii, lots of idioblasts with raphides of calcium oxalate were noticed in all tepals. The presence of idioblasts might be related to the elimination of calcium surplus from the cytosol (Paiva and Machado Bulbophyllum section Napelli (Nunes et al. Bulbophyllum species (Stpiczyńska et al. As in other orchids (Stpiczyńska et al. Bulbophyllum species could give the insights into this orchid group and likewise indicate features, which could be taxonomically important.In conclusion, the examined species, besides the macromorphological similarities, greatly vary on anatomical and ultrastructural level. More studied


Amomum compactum is one of the basic species of the traditional herbal medicine amomi fructus rotundus, with great pharmacology effect. The system position of A. compactum is not clear yet, and the introduction of this plant has been hindered by many plant diseases. However, the correlational molecular studies are relatively scarce.A. compactum cp genome and genome comparsion with three Zingiberaceae species was analyzed by corresponding softwares. Additionally, phylogenetical trees were reconstructed, based on the shared protein-coding gene sequences among 15 plant taxa by maximum parsimony (MP) and maximum likelihood (ML) methods.The total chloroplast (cp) DNA was extracted according to previous studies, and then sequenced by 454 GS FLX Titanium platform. Sequence assembly was complished by Newbler. Genome annotation was preformed by CPGAVAS and tRNA-SCAN. Then, general characteristics of the A. compactum cp genome with a classic quadripartite structure, consisting of a pair of reverse complement repeat regions (IRa/IRb) of 29,824 bp, a large single copy  region as well as a small single copy  region, is 163,553 bp in total size. The total GC content of this cp genome is 36.0%. The A. compactum cp genome owns 135 functional genes, that 113 genes are unique, containing eighty protein-coding genes, twenty-nine tRNA (transfer RNA) genes and four rRNA  genes. Codon usage of the A. compactum cp genome is biased toward codons ending with A/T. Total 58 SSR loci and 24 large repeats are detected in the A. compactum cp genome. Relative to three other Zingiberaceae cp genomes, the A. compactum cp genome exhibits an obvious expansion in the IR regions. In A. compactum cp genome, the ycf1 pseudogene is 2969 bp away from the IRa/SSC border, whereas in other Zingiberaceae species, it is only 4–5 bp away from the IRa/SSC border. Comparative cp genome sequences analysis of A. compactum with other Zingiberaceae reveals that the gene order and gene content differ slightly among Zingiberaceae species. The phylogenetic analysis based on 67 protein-coding gene sequences supports the phylogenetic position of A. compactum.The A. compactum cp genome which would be helpful for us to understand the cp genome evolution and offer useful information for phylogenetics and further studies of this traditional medicinal plant.The study has identified unique features of the The online version of this article (10.1186/s13020-018-0164-2) contains supplementary material, which is available to authorized users. Chloroplasts can provide necessary energy for plants growth as photosynthetic organelles, which also participate in other major life activities such as starch storage, sugar synthesis and many critical biological metabolic pathways. As circular DNA molecules, cp genomes mainly vary from 120 to 160 kb in size with a typical quadripartite organization in angiosperms , 4. OwinAmomum compactum  is one of the basic species of the traditional Chinese medicine amomi fructus rotundus, which is mainly produced in Vietnam and Thailand and is cultivated as a medicinal plant in the Guangdong, Guangxi and Yunnan provinces of China with great pharmacology effect. However, bacterial wilt, damping-off, leaf spot and other major plant diseases have become a severe obstacle for the introduction of this plant. Many plants belonging to the Zingiberaceae family are used as important seasoning and medicinal plants, such as Zingiber officinale, Amomum villosum, Curcuma longa, Zingiber mioga, Elettaria cardamomum, and Alpinia officinarum. In addition, previous studies have shown that the efficacy, chemical composition and pharmacological effects among the five genera of Zingiberaceae are strongly correlated. It is of great significance and broad interest to investigate the genetic relationships of traditional Chinese medicinal plants to find alternative medicinal plants. With the number of whole cp genomes in the Zingiberaceae increasing, the cp genome sequences of other species in Zingiberaceae are becoming easier to be assembled. However, studies of amomi fructus rotundus are scarce both inside and outside China, especially molecular studies.A. compactum cp genome for the first time. And to reveal the structure of this cp genome, we compare the organization (IR expansion/contraction and divergent regions) of complete cp genomes between A. compactum and other Zingiberaceae species. We also provide the result of phylogenetic analyses on basis of 67 protein-coding gene sequences from A. compactum and 14 monocot cp genomes.This study reports the assembly, annotation and structural analysis of A. compactum leaves were acquired from cultivated bases in Guangdong Province, China. The total cp DNA was extracted from roughly 100 g of leaves through an improved method by Li et al. [Fresh http://www.herbalgenomics.org/0506/cpgavas) [http://lowelab.ucsc.edu/tRNAscan-SE/). [http://ogdraw.mpimp-golm.mpg.de/) [A. compactum was submitted to NCBI GenBank database (Accession Number: MG000589).Preliminary gene annotation of this cp genome was performed by CpGAVAS, a program available online (cpgavas) . The poscpgavas)  after alan-SE/). . Then, tmpg.de/) . Finallyhttp://pgrc.ipk-gatersleben.de/misa/), with following thresholds: ten, six, five, five, five, and five repeat units for mono-nucleotide, di-nucleotide, tri-nucleotide, tetra-nucleotide, penta-nucleotide, and hexa-nucleotide SSRs, respectively. To analyze the repeat structure, REPuter [http://bibiserv.techfak.uni-bielefeld.de/reputer/) was performed to detect forward (direct) and palindromic (inverted) repeats in the cp genome. The minimum repeat unit was set to 30 bp in length, the identity of repeats was set to > 90%, and the Hamming distance equals three. All identified results were verified and redundant repeats were manually removed.Relative synonymous codon usage (RSCU) values, which were used to research the features of variations in synonymous and nonsynonymous codon usage by disregarding the composition impact of amino acid, were determined using MEGA 6.0 . Additio REPuter  , Curcuma roscoeana (KF601574), and Zingiber spectabile (JX088661), were used for comparative analysis by mVISTA program (http://genome.lbl.gov/vista/index.shtml) [A. compactum was set as the reference.Pairwise alignments of several cp genome sequences were conducted by MUMmer , and thex.shtml)  in ShuffA. compactum, 14 complete chloroplast genomes were downloaded from NCBI. The 67 shared protein-coding gene sequences were extracted using a Python script and aligned separately by ClustalW2. Phylogenetical trees were reconstructed based on 67 concatenated protein-coding gene sequences by MP and ML methods. The best-fitting model was filtrated by jModelTest 2.1.7 through the Akaike information criterion (AIC) [http://www.phylo.org/) with default parameters. Based on APGIII, Fritillaria cirrhosa was set as an outgroup. Both MP and ML analyses used 1000 bootstrap replicates.To examine the phylogenetic position of on (AIC) . The MP on (AIC)  with a hThe Minimum Standards of Reporting Checklist includes details of the experimental design, statistics, and resources used in this study.A. compactum is 163,553 bp in length with a obvious quadripartite structure  include two introns .As shown in Fig. As Table . Among tA. compactum cp genome, two genes use alternatives to ATG as start codon, as following: ATC for ndhD and ATA for rpl2. Relative synonymous codon usage (RSCU) is a statistics of uneven usage of synonymous and nonsynonymous codons in the coding sequences. An RSCU value < 1.00 indicates that the use of a codon is less frequent than expected, whereas a codon used more frequently will attain an RSCU value > 1.00. A total of 96.7% (29/30) of preferred synonymous codons, i.e., RSCU values > 1, end with A/U, whereas 90.6% (29/32) of non-preferred synonymous codons, i.e., RSCU values < 1, end with G/C. This codon usage pattern is similar with other reported cp genomes [The protein-coding gene sequences are 79,701 bp in length, which comprise 26,567 codons. And the usage frequency of codon was counted and exhibited in Table  genomes , 22, whiA. compactum cp genome  regions. A minute comparison of junctional regions between the IR and SC boundaries among C. flaviflora, C. roscoeana and Z. spectabile) were selected for comparison with A. compactum. Pairwise cp genome alignments between A. compactum and other three cp genomes regained a high degree of synteny  in the ML tree. These phylogenetic results strongly support the position of A. compactum and provide some helpful hints about relationships within the order Zingiberales.Cp genomes are widely employed in the study of evolution through phylogenetics. To examine the phylogenetic position of A. compactum, which reveals that the cp genome of A. compactum shares a quadruple structure, gene order, GC content, and codon usage features, similar to those of other land plant cp genomes. This Amomum cp genome was compared with three available Zingiberaceae cp genomes, while the genome structure and composition are similar. Also phylogenetic analysis provides new insight into phyletic evolution of this genus. Our research will contribute to species identification and evolutionary mechanisms required for the further study of A. compactum.The research assembled, annotated and analyzed the whole cp genome of Additional file 1. Minimum Standards of Reporting Checklist.Additional file 2: Table S1. Size comparison of A. compactum cp genomic regions with those of 3 other Zingiberaceae cp genomes.Additional file 3: Figure S1. Chloroplast genomic alignment between A. compactum and C. flaviflora (KR967361).Additional file 4: Figure S2. Chloroplast genomic alignment between A. compactum and C. roscoeana (KF601574).Additional file 5: Figure S3. Chloroplast genomic alignment between A. compactum and Z. spectabile (JX088661).


Polypedates leucomystax complex presents the challenging task of inferring its biogeographical history. Here, we describe its dispersion and the genetic relationships among different populations in Thailand, where we connect the population of the P. leucomystax complex of the Sunda Islands to the Indochina (mainland) population based on analyses of 266 sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Our maternal genealogy implies that there are four well-supported lineages in Thailand, consisting of Northern A (clade A: Polypedates sp.), Nan (clade B: P. cf. impresus), Southern (clade C: P. cf. leucomystax) and Northern D , with Bayesian posterior probability >0.9. Phylogeny and haplotype networks indicate that clades A, B and D are sympatric. In contrast, clade C (P. cf. leucomystax) and clade D  are genetically divergent due to the geographical barrier of the Isthmus of Kra, resulting in an allopatric distribution. Climatic conditions, in particular differences in rainfall on each side of the Isthmus of Kra, may play an important role in limiting the immigration of both clades. For the within-populations of either clades C or D, there was no significant correlation between geographic and genetic distance by the isolation-by-distance test, indicating intraspecific-dispersal of each clade. Population expansion occurred in clade C, whereas clade D showed a constant population. Taken together, the P. leucomystax complex in South East Asia may have diversified under climatic pressure, leading to allopatric and/or sympatric speciation.Taxonomic uncertainty of the Asian tree frog Microhyla fissipes  altered the topology and environmental conditions, resulting in an initial fragmentation of habitat. These mechanisms generated the high species richness observed today . Of intefissipes , Stauroiilinguis , Microhya ornata  and Polycomystax . AccuratP. leucomystax  complex, is an Asian Rhacophoridae frog. These species are widely distributed in Southeast Asia, South China and India. In addition, this species has phenotypic plasticity and exhibits high adaptation to the local environment, leading to its existence in diverse habitats such as forests and even buildings. These high levels of phenotypic plasticity present a great challenge for classification. Phylogenetic and taxonomic relationships of the P. leucomystax complex throughout Southeast Asia exhibit adaptive radiation  and then sequenced .A total of 266 adult Thailand . All samCOI sequences of the P. leucomystax complex that were retrieved from GenBank: P. impresus: KP996822 (China),  KP996846 (China),  KP087862 – KP087870 (Laos); P. leucomystax:  KR087871 – KR087872 (Thailand); and P. megacephalus:  KR087879,  KR087881 (Thailand). First, the best-fit model of DNA sequence evolution for this locus was identified with the Akaike information criterion (AIC) implemented in MrModeltest v2.3 (Bayesian inference (BI) and maximum likelihood (ML) were employed for constructing a phylogenetic tree based on the following partial est v2.3 , resultiest v2.3  with twoest v2.3  and builest v2.3  using thCOI were aligned using ClustalW (implemented in MEGA 6.0 with default parameters). The number of polymorphic sites, the parsimony-informative sites, singleton sites, the number of haplotypes, haplotype diversity (Hd), and nucleotide diversity for each clade were calculated using DnaSP v5.0  of the observed distribution was calculated . After multiple alignment of all COI sequences, the sequences were trimmed to the same length (437 bp) before downstream analysis. We observed 82 polymorphic sites, which are also 82 parsimony-informative sites without a singleton site, resulting in the acquisition of 15 haplotypes , Polypedates sp.; clade B (Nan), P. cf. impresus; clade C (Southern), P. cf. leucomystax; and clade D (Northern D), P. cf. megacephalus , MHS, KCB, PCB, Saraburi (SRB), Loei (LPR), Nakhon Ratchasima (NRS) and Prachuap Khiri Khan (PKK). The dispersal areas of clade C, as represented by P. cf. leucomystax, included Chumphon (CP), Nakhon Si Thammarat (NST), Phuket (PK) and Ranong (RN), which are south of the Isthmus of Kra   shared haplotype J. Haplotypes F and G of clade D are considerably divergent from the rest of clade D and we partition it in two subclades: D1 and D2. Clade C (P. cf. leucomystax) had high haplotype diversity and contained three unique haplotypes  and two shared haplotypes (K and L).A minimum spanning network among the mitochondrial haplotypes was also constructed as shown in  0.7526) . HaplotyCOI gene sequence and the geographical distance found no significant isolation-by-distance effect among populations of clade C (P. cf. leucomystax) and clade D   .P. cf . leucomystax) and clade D , neutrality tests (Tajima’s D and Fu’s Fs) of both species were not significantly positive, whereas Fu’s Fs of clade C was significantly positive (rg (0.2031) but a unimodal mismatch distribution for clade C with a low rg (0.0569) (When we defined a significant barrier around the Isthmus of Kra (IOK) leading to the genetic divergence between clade C (positive . Further(0.0569) . OverallP. leucomystax complex is contentious due to the species’ widespread distribution from Nepal to South East Asia and similar morphologies. To better understand the population structure and biogeography of the P. leucomystax complex in Thailand, where there is a substantial area for their genetic dispersal, the COI mitochondrial gene sequences of these species were analysed. Our matrilineal genealogy implied four well-supported lineages, consisting of a Northern D clade (clade D), a Southern clade (clade C), a Nan clade (clade B) and a Northern A clade (clade A); based on their sequences and distributions, they might be treated as P. megacephalus, P. leucomystax, P. impresus and Polypedates sp., respectively.The taxonomy of the Asian tree frog of the Triturus) form a hybrid zone (south of the Marmara Sea), where introgression suggests movement of the hybrid zone (P. leucomystax complex (P. leucomystax complex from Indochina shared nuclear brain-derived neurotrophic factor (BDNF) and recombination activating gene 1 (RAG-1) haplotypes with one sample from the south of IOK, possibly suggesting deep coalescence or incomplete lineage sorting.Although the mtDNA phylogeny reveals the presence of distinct clades, mtDNA introgression is frequently observed where closely related species are in secondary contact . This carid zone . Therefo complex . KuraishP. leucomystax complex in Southeast Asia and revealed that P. megacephalus is restricted to Indochina    to hypothesize that the existence of a seaway across the IOK over a million year contributed to its geographically discontinuous distribution. In contrast, P. megacephalus (putative Northern D clade) and P. leucomystax (putative Southern clade) was estimated to be in the late Pliocene or early Pleistocene (1.4–4.0 MYBP) (P. leucomystax) expanded and colonized the northern part of Indochina, leading to the diversification of the species. This hypothesis is partially supported by the results of the unimodal pairwise difference and the small value of the raggedness index in the Southern clade  corresponds to a considerable phylogeographic break as evidenced by the large genetic divergence between the populations on either side of the isthmus. n China) , while tipelago) . Based o.0 MYBP) . We assurn clade .P. megacephalus is required. According to the demographic history, the population of Northern D was a stable population, but the population of Southern clade showed a relatively similar unimodal distribution with a small raggedness index, possibly indicating a population expansion. This result was similar to that observed in the population of P. leucomystax in the northern Philippines  seemed to represent a naturally occurring divergence because of the emergence of endemic haplotypes; however, it was a low-supported lineage, with 0.7 Bayesian posterior probability (BPP). When we considered the topography of this region, the population of NRS as clade D1 is partitioned from the other populations within clade D by the two mountain ranges, Dong Phaya Yen and Sankamphaeng. This may be a possible barrier to gene flow among the modern populations of Northern D between eastern (clade D1) and western  Thailand. Unfortunately, only a limited number of populations from the east of Thailand were investigated in this study. Further work for validating whether Dong Phaya Yen and Sankamphaeng are a barrier to gene flow in lippines . This scP. leucomystax complex in Thailand suggested four lineages, i.e., Nan (putative P. impresus), Northern D , Southern (putative P. leucomystax) and Northern A (Polypedates sp.) clades. We noted that the populations of the Northern D, Nan and Polypedates sp. clades are in sympatry, while their distributions are allopatric to the southern clade (P. leucomystax) due to the separation by the Isthmus of Kra. Climatic conditions may be a major contributor to limited migration of the current populations of both clades, but climatic oscillation in the Pliocene and Pleistocene is a highly possible scenario that drove speciation resulting in diversification of the P. leucomystax complex in Southeast Asia and China, which includes the divergence of the southern and northern clades in Thailand.Our matrilineal genealogy of the 10.7717/peerj.4263/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.4263/supp-2Supplemental Information 2Click here for additional data file.


An author name was incorrectly spelled as Andrew M. Koutnik. The correct spelling is Andrew P. Koutnik. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.International Patent # PCT/US2014/031237, University of South Florida, DD, SK, P. Arnold, “Compositions and Methods for Producing Elevated and Sustained Ketosis.” P. Arnold (Savind) has received financial support (ONR N000140610105 and N000140910244) from DD (USF) to synthesize ketone esters. Provisional patent # 16A007, University of South Florida, CA, P. Arnold, DD “Exogenous Ketone Supplements for Reducing Anxiety-Related Behavior.” The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tricholoma matsutake  is a prized mushroom in Asia due to its unique flavor and pine aroma. The fruiting body of PM forms only in its natural habitat (pine forest), and little is known regarding the natural conditions required for successful generation of the fruiting bodies in this species. Recent studies suggest that microbial interactions may be associated with the growth of PM; however, there have been few studies of the bacterial effects on PM growth. In this study, we surveyed which bacteria can directly and indirectly promote the growth of PM by using co-cultures with PM and molds associated with the fruiting body. Among 16 bacterial species isolated from the fruiting body, some species significantly influenced the mycelial growth of PM and molds. Most bacteria negatively affected PM growth and exhibited various enzyme activities, which suggests that they use the fruiting body as nutrient source. However, growth-promoting bacteria belonging to the Dietzia, Ewingella, Pseudomonas, Paenibacillus, and Rodococcus were also found. In addition, many bacteria suppressed molds, which suggests an indirect positive effect on PM as a biocontrol agent. Our results provide important insights toward a better understanding of the microbial interactions in the fruiting body of PM, and indicate that growth-promoting bacteria may be an important component in successful cultivation of PM. Pseudomonas putida and button mushrooms (Agaricus bisporus) [Macrofungi develop large mycelial fruiting bodies that serve as reproductive organs producing spores , which aisporus) . Bacteriisporus) , and proisporus) .Pseudomonas tolaasii is notorious for causing brown blotch of fruiting bodies by degrading them with secreted toxins [e.g. Ewingella, Pseudomonas, and Stenotrophomonas) are known to degrade mycelia by exhibiting high chitinase activity [In contrast, bacteria can have negative associations with fruiting bodies. As pathogens and decomposers, bacteria often use fruiting bodies as a nutrient source . For exad toxins . In factactivity –13.Tricholoma matsutake, is a naturally occurring edible mushroom, highly prized in Asia for its unique flavor and pine aroma [e.g. Janthinobacterium, Pseudomonas, and Stenotrophomonas) have been found in PM fruiting bodies, and the community composition appears to be different than that of the soil bacteria found in the fairy ring of PM [The pine mushroom (PM), ne aroma . The PM ne aroma . PM fruine aroma –18. Thisne aroma –22. Recene aroma –25, suggne aroma . Severalng of PM , 27.Because bacteria can influence the physiology of mushrooms , 8, a beA total of five fresh PM fruiting bodies were collected in September, 2013 from the research forest of the National Institute of Forest Science in Hongcheon , Gangwon province, Korea . Sampling sites for each fruiting body were separated by more than 300 m. We used only fresh and immature fruiting bodies  to reducTM PCR Purification Kits  following the manufacturer’s instructions. Purified PCR products were sequenced using an ABI3700 automated DNA sequencer at Macrogen . The sequences were aligned using MAFFT [Bacteria colony PCR amplification was performed using a Maxime PCR PreMix kit . The PCR mixture contained 1.0 μl of each of the 27F and 1492R primers , 1.0 μl ng MAFFT  and editng MAFFT . Phylogeng MAFFT . Based ong MAFFT . SequencTricholoma matsutake’ Media (TMM)  which is one of the best media for the growth of PM [To prepare bacterial inocula for the co-culture experiment, bacterial strains were recovered from the stocks we isolated and stored on TSA medium at 30°C. Co-culture was performed using 60 mm Petri dishes containing ‘th of PM , 34. We Bacterial strains were co-cultured with molds to assess the antifungal activity of bacterial species. For covering various molds that present naturally in PM fruiting bodies, we used 17 isolates of mold species that were originated from PM fruiting bodies and deposited at the Seoul National University Fungal Collection (SFC) . Similar2·H2O (0.1 g/L), and 1% Tween80 [We assessed the activity of five different enzymes associated with fungal cell wall component—chitinase, cellulase (endoglucanase and β-glucosidase), protease, and lipase—for bacteria and molds. Chitinase activity was assessed based on the Roberts and Selitrennikoff method . Colloid Tween80 . The assSerratia marcescens was the most dominant species of bacteria (34.5%), followed by Mycetocola lacteus (17.3%) and Pseudomonas endophytica (16.4%). Bacterial diversity varied largely among parts of the fruiting body: the outside of the pileus harbored the largest number (10 species), while the inside of the stipe harbored the least (3 species) , while the number of bacterial species was comparable between pileus parts (P = 0.64) and between stipe parts (P = 0.98) , except inside the stipe, where Ps. endophytica was dominant (85.0%) . 16S rDNA sequence analysis for 110 bacterial isolates revealed a total of 16 different species of bacteria . All 16Sspecies) . The num = 0.98) . In a to (85.0%) .Se. marcescens, My. lacteus, and Stenotrophomonas maltophilia. In contrast, six bacterial species stimulated PM mycelial growth significantly (growth change: 123–194%)  and Ps. koreensis (growth change: 190%) had the largest growth-promoting effect  was observed when these species were present . The bac23–194%) . This grg effect . Based og effect .Ewingella americana had a suppressive effect on the widest range of mold species , followed by Se. marcescens  and Ps. endophytica . Growth response of our 17 test molds to the 16 bacteria separated into two groups: a generally suppressed group of molds (mGroup 1) and a specifically influenced group of molds (mGroup 2)  and R. degradans (113%), and growth of Umbelopsis nana was significantly increased by My. lacteus (Growth change: 113%), Pa. taichungensis (Growth change: 113%), and Staphylococcus lentus (Growth change: 116%) (Among the 272 bacteria-mold pairings (16 bacteria × 17 molds) we cultured, a change in growth was significant for 66 pairs : 61 pairGroup 2) . mGroup e: 116%) .Se. marcescens and Stenotrophomonas maltophilia, exhibited activity for all enzymes tested. Chitinase activity was found in three species. Seven species showed cellulase activity. Protease activity was detected from six species and lipase activity from seven species , ten species showed enzyme activity. Only two species,  species .Sa. kiliense and Pe. crustosum exhibited enzyme activity for all enzymes tested except chitinase. In the Ascomycota, four Penicillium species showed cellulase and protease activities. Among seven species in the Zygomycota, only two species showed enzyme activity: Absidia sp. had protease activity and U. nana had cellulase and lipase activities  [Ps. endophytica, which was first reported as an endophytic bacterium in the stem and root of potato [Agaribus bisporus [Armillaria mellea [Cantharellus spp. [Tuber borchii [Suillus grevillea [Diverse bacteria have been reported from PM fruiting bodies by culture dependent and high-throughput sequencing methods. Recently, in China . Pyroseqes of PM . Our restophilia . Althougacillus) . Althougacillus) , 39. Intf potato . Severalbisporus , Armillaa mellea , Cantharlus spp. , Tuber b borchii , and Suirevillea . This suPs. putida induces initiation of fruiting body formation in Agaricus bisporus [Streptomyces sp. and Ps. fluorescence promote hyphal growth and mycorrhizal formation in Amanita muscaria and Laccaria bicolor, respectively [Tuber melanosporum, the bacterial community is involved in fruiting body maturation [Paenbacillus, Pseudomonas, and Rhodococcus are well-known mycorrhiza helper bacteria (MHB) [Ps. endophytica . Molds could be separated into two groups according to suppression pattern: a generally suppressed group (mGroup 1) and a specifically influenced group (mGroup 2)  . The gening body , ectomycing body , and soigs of PM , 63, andgs of PM . Thus, tPa. taichungensis, Se. marcescens, and Stenotrophomonas maltophilia showed chitinase and cellulase activities as reported previously [e.g. chitinase and protease) are often responsible for the antifungal activity of bacteria [Many of the bacteria isolated from PM fruiting bodies showed high enzyme activity, which suggests mycophagy in these bacteria is associated with PM fruiting bodies, a phenomenon that has been documented in other studies , 65–67. eviously , 68, 69.eviously , high enbacteria –73. In oe.g. sugar and amino acid), rather than consuming the fungal cell wall [The common enzyme activities observed in bacteria and molds suggest that bacteria and molds compete for the same nutrient sources. Therefore, antifungal activity may be high in the bacteria-mold pairs that shared same enzyme activity. However, our results did not show any relationship between enzyme activity and antifungal activity, which suggests that bacteria may provide indirect positive effects to PM as a biocontrol agent, irrespective of nutrient competition. Bacteria often use nutrients provided from associated fungi  on the mycelial growth of PM, as well as indirect positive effects of bacteria as biocontrol agents against molds. The mechanisms associated with these bacterial effects, i.e. specific metabolites and optimal growth conditions, remain to be elucidated and a better understanding of these factors may lead to successful cultivation of PM.In conclusion, while many bacterial species have a negative effect on the growth of PM mycelia and likely exploit the fruiting body of PM as a nutrient source, others have a positive influence on PM growth. We found direct positive effects of bacteria (S1 Table(DOCX)Click here for additional data file.S2 TableSignificance of difference was tested using pairwise t-test adjusted by the false discovery rate of Benjamini and Hochberg. Significantly different growth changes are highlighted in grey. Significant growth promotions are indicated by bolded numbers.(DOCX)Click here for additional data file.


Escherichia coli (E. coli) urinary tract infections (UTIs) over five years, from 2009 to 2013, and compares prevalence of resistance in hospital- and community-acquired E. coli UTI. A cross sectional study of E. coli UTIs from patients attending a tertiary referral hospital in Canberra, Australia was undertaken. Time series analysis was performed to illustrate resistance trends. Only the first positive E. coli UTI per patient per year was included in the analysis. A total of 15,022 positive cultures from 8724 patients were identified. Results are based on 5333 first E. coli UTIs, from 4732 patients, of which 84.2% were community-acquired. Five-year hospital and community resistance rates were highest for ampicillin (41.9%) and trimethoprim (20.7%). Resistance was lowest for meropenem (0.0%), nitrofurantoin (2.7%), piperacillin-tazobactam (2.9%) and ciprofloxacin (6.5%). Resistance to amoxycillin-clavulanate, cefazolin, gentamicin and piperacillin-tazobactam were significantly higher in hospital- compared to community-acquired UTIs . Trend analysis showed significant increases in resistance over five years for amoxycillin-clavulanate, trimethoprim, ciprofloxacin, nitrofurantoin, trimethoprim-sulphamethoxazole, cefazolin, ceftriaxone and gentamicin  with seasonal pattern observed for trimethoprim resistance . An association between ciprofloxacin resistance, cefazolin resistance and ceftriaxone resistance with older age was noted. Given the relatively high resistance rates for ampicillin and trimethoprim, these antimicrobials should be reconsidered for empirical treatment of UTIs in this patient population. Our findings have important implications for UTI treatment based on setting of acquisition.This study describes the antimicrobial resistance temporal trends and seasonal variation of  Escherichia coli (E. coli), a Gram negative bacillus [Urinary tract infections (UTIs) are predominantly bacterial infections affecting people both in the community and in hospitals . Over 80bacillus . Data frbacillus . Visits bacillus . In Austbacillus .E. coli is increasing [E. coli specimens obtained from a university hospital from 1997 to 2007 found an increasing trend in resistance to trimethoprim/sulfamethoxazole, ciprofloxacin and amoxycillin/clavulanic acid  [E. coli strains resistant to beta-lactam antibiotics and ciprofloxacin [E. coli isolates to ampicillin and ciprofloxacin increased from 48% to 51% and 8% to 11% respectively [E. coli to various antimicrobials show large inter-country variability [E. coli resistance rates differ for hospital-acquired and community-acquired UTIs [While UTIs are a major infection burden globally, the growing problem of antimicrobial resistance (AMR) can result in treatment failures and increased cost of healthcare . There icreasing . In Switctively) . The Ausred UTIs . CompariE. coli resistance levels is not completely known in Australia. Obtaining this information is important because it not only provides knowledge about the health status of a population, but also contributes to disease management decisions [E. coli UTI over five years at an Australian tertiary hospital. The study also compares the prevalence of resistance between hospital- and community-acquired E. coli UTIs.The prevalence of AMR including hospital and community urinary ecisions . This stA retrospective cross sectional design was used. The study was conducted with data from ACT Pathology which is based at a tertiary referral hospital, the Canberra Hospital and Health Services. This is Australian Capital Territory’s (ACT) main hospital which provides acute and specialist care services to over 600,000 people in the surrounding region. The 600 bed publicly-funded hospital which includes an emergency department and intensive care unit, offers a comprehensive range of health services such as acute inpatient and day services, outpatient services, women's and children's services and pathology services. Solid organ transplant services are not offered in Canberra.Human research ethics approval was granted by ACT Health Human Research Ethics Committee’s Low Risk Sub-Committee and Australian Catholic University Human Research Ethics Committee. Consent from patients was not obtained as a waiver of consent was granted by the ethics committees.The microbiology records of inpatients and those attending Canberra Hospital who had urine samples processed at ACT Pathology from January 2009 to December 2013 were retrospectively reviewed. Demographic data and clinical information such as date of birth, gender, admission date, specimen collection date and antimicrobial susceptibility test result were obtained from the microbiology laboratory database and administrative record system.7 colony forming unit (cfu) per litre of urine was considered positive for UTI based on the laboratory recommendations. This 107 cfu/L cut-off is commonly used as it increases the sensitivity of the urine culture test making it a practical threshold [E. coli [E. coli UTI per patient per year was included in the final analysis.Urine samples were analysed and processed based on the microbiology laboratory standards . For thihreshold . The cri[E. coli ,17. CultE. coli urine cultures obtained within the first 48 hours of admission (including cultures from non-admissions such as outpatient clinics) were defined as community-acquired UTIs. Positive cultures obtained more than 48 hours after admission and within 48 hours of discharge were defined as hospital-acquired UTIs.Urine cultures were classified based on the setting of acquisition of infection  using criteria from the Centers for Disease Control and Prevention definitions . PositivE. coli (>107cfu/L) isolated after overnight incubation on culture had disc susceptibility testing done. The antibiotic discs used for these tests were ampicillin (10μg), amoxycillin-clavulanate (augmentin) (30μg), cephalexin/cefazolin (30 μg), trimethoprim (5μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), nitrofurantoin (300 μg) and gentamicin (10μg). The isolates which were found to be resistant to at least three of the routinely tested antibiotics were then sent for Vitek2 testing to determine the MICs for ceftriaxone, trimethoprim-sulphamethoxazole, meropenem and piperacillin-tazobactam in addition to the routinely tested antibiotics. Direct susceptibility testing method on urine specimens for E. coli has been validated at ACT Pathology and is comparable to the CLSI recommended methods.Antimicrobial susceptibility was performed by a disc diffusion method and the automated minimum inhibitory concentration (MIC) method using Vitek2 (Biomerieux Diagnostics). Interpretation was based on Clinical Laboratory Standard Institute  criteria . Based oE. coli ATCC 25922, E. faecalis ATCC 29212 and for Vitek E. coli ATCC 25922, P. aeruginosa ATCC 27853, E. faecalis ATCC 29212, S. aureus ATCC 29213.The quality control strains used for disc diffusion tests were K. pneumoniae ATCC 700603 was used as the quality control strain.Detection of ESBL-producing isolates was performed with combination discs of cefotaxime (30μg), cefotaxime/clavulanic acid (30/10μg), ceftazidime (30μg) and ceftazidime/clavulanic acid (30/10μg) whenever required according to CLSI guidelines . ExtendeE. coli to the routinely tested first-line antimicrobials on over 4,000 isolates  were calculated by dividing the number of urinary E. coli isolates resistant to each antimicrobial by the number of isolates tested against an individual antimicrobial agent. For the isolates which were sent for further susceptibility testing on Vitek2 against second-line antimicrobials , the denominator used in calculating the resistance rates was the total number of isolates included in the study. This denominator was used based on the assumption that isolates were initially not tested for the Vitek2 antibiotics because they were considered highly unlikely to be resistant to these antibiotics. Hence in order not to overestimate the resistance rates of these isolates the denominator included all isolates tested on both antibiotic discs and Vitek2. The binomial exact 95% confidence intervals (CI) of the resistance percentages were calculated. The 5-year resistance rates were compared for community- and hospital-acquired isolates. The chi-square test was used to check for statistically significant differences in AMR between both groups. Mean differences in age between the two groups were tested using Student’s t-tests. A time series analysis was performed separately for all antimicrobials tested to identify patterns in resistance  over the five year period. Seasonality is a pattern that shows periodic repetitive fluctuations over time. An autoregressive (AR) model was constructed to assess time-varying resistance patterns  and multiple time series models were fitted to also account for age and sex. The analysis on age and sex followed an ecological study design because these variables were aggregated for each season. The Dickey-Fuller (DF) and the augmented Dickey-Fuller (ADF) tests were used to assess a unit root in the time series data. Both DF and ADF statistics are negative numbers; the more negative, the stronger the rejection of the null hypothesis (that there is unit root at some level of confidence). These unit root tests investigate whether a time series variable  is non-stationary using the AR model [E. coli isolates for which the antimicrobial showed an intermediate susceptibility category  were excluded from the final analysis. A significance level of P < 0.05 was used. Data were analysed using STATA statistical software .The overall 5-year and yearly resistance rates of AR model . UrinaryE. coli being the most common organism isolated in 7670 (51.1%) samples. The distribution of samples by study year is shown in A total of 106,512 urine samples from 47,727 patients attending Canberra Hospital from 2009 to 2013 were processed by ACT Pathology. Of these, 14.1%  had positive cultures with E. coli cultures, most (7103 isolates) could be further classified as community- or hospital-acquired UTI based on available data. The data were then restricted to the first positive E. coli UTI per patient per year of which there were 5346 positive E. coli UTIs but only 5333 had susceptibility test results. Hence 5333 E. coli UTIs belonging to 4732 patients in the 5-year period were included in the final analysis. The majority  of UTIs were classified as community-acquired and 15.8% (n = 841) as hospital-acquired. The mean age of all patients was 57.0 years (SD = 27.5) and patients were mostly female . There was a significant difference in age between patients with hospital- and community-acquired E. coli UTI  but no significant differences in gender.Of the 7670 All 5333 isolates had routine susceptibility testing performed against first-line antimicrobials and the overall 5-year and stratified  AMR rates are summarised in E. coli for both hospital and community isolates combined were seen for ampicillin  and trimethoprim . The lowest resistance rates were for meropenem (0.0%), nitrofurantoin  and piperacillin-tazobactam . Resistance to amoxycillin-clavulanate, cephalexin/cefazolin, gentamicin and piperacillin-tazobactam was significantly higher in hospital- compared to community-acquired UTIs . For ampicillin, trimethoprim, nalidixic acid, ciprofloxacin, ceftriaxone and trimethoprim-sulphamethoxazole, resistance rates were also higher for hospital- compared with community-acquired UTI but this did not reach statistical significance . Extended spectrum beta-lactamase production was low by international standards but was significantly higher in hospital-acquired  compared with community-acquired UTIs . The levels of ESBL-producing E. coli increased from 0.7% (95% CI = 0.0–3.8) in hospital-acquired UTIs in 2009 to 6.5% (95% CI = 3.2–11.6) in 2013. An increase was also noted for community-acquired UTIs . The increasing trend in ESBL production over the five years was statistically significant for both hospital (P = 0.035) and community-acquired UTIs (P<0.001).Overall 5-year prevalence of ESBL-producing E. coli UTIs in an Australian tertiary hospital. To our knowledge this is the first Australian study to compare AMR in hospital- and community-acquired E. coli UTI and assess AMR temporal trends and seasonal variation of E. coli UTI over time. Our results showed that overall resistance was highest for ampicillin and trimethoprim. We also found significantly higher resistance rates in hospital- compared to community-acquired UTIs for amoxycillin-clavulanate, cephalexin/cefazolin, gentamicin and piperacillin-tazobactam with an increasing resistance trend for eight of the twelve antimicrobials tested which include the four commonly used antimicrobials for first line treatment of UTI in Australia.This study provides information about the AMR pattern of E. coli compared to most countries [In Australia, trimethoprim, cephalexin, amoxycillin-clavulanate or nitrofurantoin are recommended for first line treatment of UTI . The Infountries . Our oveountries ,24, demoountries . The expountries . It was ountries ,27. Seveountries –30. HencE. coli UTIs, consistent with other studies [E. coli, a bacterium carried in the bowel and acquired in the community, is brought into hospital usually by patients themselves rather than being hospital-acquired. This finding may also have been partially dictated by our methodology from using the first positive UTI per person per year. The different resistance rates for hospital- and community-acquired urinary E. coli isolates seen in this study are comparable with findings reported previously [E. coli in Canberra [E. coli resistance rates, antibiograms should be stratified by setting of infection onset [The sample size of the data was able to detect some significant differences between community- and hospital-acquired UTI resistance rates but for some antimicrobials the differences observed could not be confirmed statistically, possibly due to an insufficient sample size. Overall, we found lower rates of antibiotic resistance for community- compared with hospital-acquired  studies ,31. The eviously ,11. SimiCanberra . While tCanberra . We agreon onset .The increasing resistance trend noted in our study for the eight antimicrobials is consistent with previously reported Australian data and published studies from other developed countries ,9,35,36.E. coli UTI [E. coli in both hospital and community-acquired UTIs pose considerable public health concern. This is because this organism renders many of the conventional empirical treatment options for UTI ineffective especially in community-acquired UTI where options for oral antibiotic therapy appear to be limited [E. coli, carbapenems are considered the treatment of choice [It is worth emphasizing that our overall ESBL rate of 1.9% was low compared to most other published studies . Resultscoli UTI . Althoug limited . For hosf choice . In our E. coli UTI per person per year may have underestimated the resistance rates reported in our study. Evidence suggests that analysis of antimicrobial resistance data should include each individual positive isolate in order to ensure sensitivity, but this positive isolate should only be included once to guarantee specificity [E. coli [This study has some limitations. As most UTIs are treated empirically, it is possible that samples submitted to the laboratory included patients with recurrent UTIs and asymptomatic bacteriuria thereby overestimating the resistance rates. In addition, inclusion of the first positive cificity . This ap[E. coli ,34. It i[E. coli  there isE. coli to commonly prescribed antimicrobials. Amoxycillin-clavulanate and nitrofurantoin are still effective for empirical treatment of UTI in this population. Overuse of ampicillin and trimethoprim should be avoided given the high resistance rates reported. In developing local antimicrobial prescribing guidelines, the choice of antimicrobial in the treatment of UTI should be based on setting  of acquisition.Antimicrobial resistance poses grave concerns for antimicrobial effectiveness in treating infections such as UTI. This study demonstrates the increasing resistance of urinary S1 Table(DOC)Click here for additional data file.


Harmonia axyridis strain. The combined action of ladybirds and hymenopteran parasitoids could have a net positive effect on aphid population suppression and, consequently, on host (crop) plants. However, ladybird encounters with aphid-tending or foraging ants must be reduced. Deploying ladybirds to help manage aphids in greenhouses and similar protective structures is encouraged.As part of my research on the mass production and augmentative release of ladybirds, I reviewed the primary research literature to test the prediction that ladybirds are effective aphid predators in greenhouses. Aphid population reduction exceeded 50% in most studies and ladybird release rates usually did not correlate with aphid reduction. The ratio of aphid reduction/release rate was slightly less for larvae than adults in some studies, suggesting that larvae were less effective (than adults) in suppressing aphids. Some adult releases were inside cages, thereby limiting adult dispersion from plants. Overall, the ratio of aphid reduction/release rate was greatest for ladybird adults of the normal strain , but least for adults of a flightless  Culturing plants in greenhouses, glasshouses, or hothouses has existed in Europe and Asia since the early 19th century, with expansion into North America and other regions of the world in recent years ,3. DespiAphis gossypii Glover, Macrosiphum euphorbiae (Thomas), Aulacorthum solani , and Myzus persicae Sulzer  at post-release evaluation; (2) In the absence of controls, or if aphid density was variable at the onset of the experiment, percent aphid reduction was calculated from aphid density on plants before releasing ladybirds (pre-release) and after releasing them (post-release) [(pre-release − post-release)/pre-release (×100)].t-test compared the ratio of aphid population reduction/release rate between larvae and adults. Correlations and mean values were significantly different when p < 0.05. SigmaStat 3.0.1  software assisted with data analysis.To compare ladybird efficacy across studies listed in Vicia faba L. 79S4 cultivar  reduced the reproductive rate of the black bean aphid Aphis fabae Scopoli in pots enclosed in cages [A. fabae herbivory. Allelochemicals are secondary plant compounds that are sometimes toxic to aphids, and thereby reduce herbivory. These compounds may or may not affect predation capacity of ladybirds which consume aphids on defended plants. A release ratio of 1:1  per plant, reduced aphid density by 57.1% on the partially resistant cultivar, but just 33% on the susceptible cultivar  in nine days, when compared against control plants, caged plants without ladybird beetles  on caged pea Pisum sativum L. plants with a reduced leaf wax phenotype than on those with a normal leaf wax phenotype [H. convergens on plants in cages, thereby reducing its effectiveness as an aphid predator.Morphological plant defenses, such as the degree of “waxiness” on the leaf surface, can affect predation potential of ladybirds foraging on plants. For example, adults of the convergent lady beetle henotype . Waxy leH. convergens) spent less time foraging on potted wild potato Solanum berthaultii Hawkes containing a high density of glandular trichomes on its leaves and stems than on an interspecific hybrid of cultivated potato Solanum tuberosum L. x wild potato S. berthaultii F3, containing a moderate density of glandular trichomes, or on S. tuberosum, containing no glandular trichomes [H. convergens predation capacity was presumably reduced on potato foliage containing high trichome density.Plant epidermal hairs  can alter the foraging behavior of coccinellids on plants and are known to impale or trap some aphid species . Trichomrichomes . Thus, HColeomegilla maculata DeGeer did not remain on potted cucumber Cucumis sativus L. plants long enough to suppress densities of the melon/cotton aphid A. gossypii [C. sativus leaves were perceived as being “irritating” to C. maculata larvae. The effect of trichomes on ladybird larvae is species specific. In the same study, C. sativus glandular trichomes did not negatively affect predation capacity of 2nd instar larvae of other ladybirds, such as Cycloneda sanguinea (L.) and Adalia bipunctata (L.). Aphid populations were reduced by 50.4% and 36.0% in 11 days by C. sanguinea and A. bipunctata, respectively. Thus, cucumber trichomes have differential effects on ladybird larvae; C. maculata were affected negatively, but C. sanguinea and A. bipunctata were not.A reduction in foraging time on plants containing trichomes can result in a reduction of aphid control, as shown for 2nd instar larvae. Note that 2nd instar larvae of the ladybird gossypii . GlandulGossypium hirsutum L., representing plants with glabrous (low), hirsute (moderate), and pilose (high) trichome density, did not reduce the predation capacity of C. sanguinea and H. convergens [A. gossypii) and two days later, a single H. convergens or C. sanguinea adult female was released onto infested plants. H. convergens and C. sanguinea females reduced the aphid population on plants by an average of 87% and 93.5%, respectively, regardless of trichome density and within two days of release, in comparison to aphid density on plants not inoculated with a ladybird.The Antares, CNPA 7H, or DeltaOpal cultivars of cotton nvergens . Young pHost plant defenses), aphid population reduction did not correlate with release rate ; and ladybird larvae were not significantly less effective than adults, based on a ratio of aphid reduction/release rate , 2nd instar larvae of three species, C. maculata, A. bipunctata, and C. sanguinea reduced the density of M. persicae [M. persicae per pot (four plant cuttings per pot), the density of aphids was reduced by 97%, 98%, and 99% in seven days, due to predation by C. maculata, A. bipunctata, and C. sanguinea, respectively  and sweet pepper (Capsicum annuum L.), respectively, in 8–10 days at several release rates. The release rate (number of ladybirds/ number of aphids) did not appear to have an effect on aphid reduction.In another study, 9.0 mpunctata . A singlLemnia biplagiata (Swartz) and Leis dimidiata (F.), were tested for their capacity to reduce melon aphid (A. gossypii) numbers on cucumbers [L. dimidiata were more effective than adults, since adults tended to disperse from plants at low aphid density. The larvae continued to search foliage for aphids, even at low aphid density. For L. dimidiata, a release ratio of 1:10 or 1:20, ladybird: aphid was suitable to suppress A. gossypii populations by 85%–90%. Unfortunately, the authors didn’t provide information on A. gossypii density at release or post-release of L. dimidiata. Despite this, the authors recommend using both L. dimidiata and L. biplagiata to suppress aphid populations on cucumbers, peppers, and eggplants [In a preliminary study, two ladybirds, ucumbers . The autggplants .Harmonia axyridis  larvae (274 3rd and 4th instars) and adults (726 overwintered adults) were released on strawberry (Fragaria x ananassa Duchesne) plants to reduce populations of two aphid species, Aphis forbesi Weed and Chaetosiphon minor (Forbes) [H. axyridis larvae (750 3rd and 4th instars) and no adults were released onto strawberry plants. Aphid density decreased over 29 days from approximately 1300 to 200 aphids per 20 plants as a result of H. axyridis predation, suggesting that many of the larvae remained on the plants, fed on aphids, and pupated successfully; emerging adults contributed to aphid control as well. Note that the initial release rate was high (0.58 larvae/aphids), the time frame of the study was 29 days, and aphid reduction was 85%. Apparently, newly emerged H. axyridis adults were prevented from exiting the greenhouse.(Forbes) . In one 3 nylon) cages, C. maculata 3rd instars showed promise in reducing the cotton aphid, A. gossypii, particularly at high population densities [C. maculata, A. gossypii densities were reduced by 87% and 96%, at a release rate of 0.20 and 0.33 ladybird/aphid density, respectively. At a lower release rate (0.07 ladybird/aphid), the A. gossypii population was reduced by 47%.In a study involving aphids on strawberry plants, in replicate  to suppress A. gossypii and Aphis craccivora Koch on sweet pepper (C. annuum). Although the quantity or life stage of the ladybirds released was not specifically mentioned, the authors suggest that both ladybird species contributed to aphid population reduction in the test greenhouse in contrast to the control greenhouse (without ladybirds). Because of the lack of ladybird release rates, this study was not listed in In another study, y plants . The prand fruit . Neithernd fruit  releasedM. persicae was reduced significantly by H. axyridis (presumably adults) on chile Capsicum annum L. plants held in screened cages [H. axyridis and M. persicae at ladybird: aphid ratios ranging from 1:20 to 1:640 in replicated treatment cages (see H. axyridis), aphid density was significantly reduced by at least 95% at all densities, except the 1:640 density (85.7%), over a 10-day sampling period. This study shows the potential of H. axyridis adults as a predator of M. persicae on chile plants. It is very unlikely that the same positive results would be achievable if chile plants were not enclosed in cages, because of the propensity of H. axyridis adults to fly away from plants, as documented in other studies.The population density of ed cages . In the ages see . In compCucumis sativus L.) plants artificially infested with A. gossypii at a density of 50 aphids per plant were exposed to neonate larvae (emerging from egg clutches) of H. axyridis of a lab-cultured flightless strain, in replicate greenhouses, including a control greenhouse of identical dimensions (but no ladybird releases) [A. gossypii within five days post-release. By the eighth day, A. gossypii began to increase their densities again, suggesting that multiple releases of H. axyridis egg clutches and/or 1st instar larvae would be necessary to further reduce the aphid population.Cucumber (eleases) . ReleaseH. axyridis were tested as biological control agents against M. persicae and turnip aphid Lipaphis erysimi  on seedlings of non-heading Brassica rapa L. (2 cultivars) [H. axyridis adults per m2 or 10 larvae per m2 area of plot (80 plants/plot) were sufficient to prevent significant increases in aphid densities over three weeks. In comparison to control plots (no releases), M. persicae density was 72%–98% lower and L. erysimi density was 81% lower in test plots with one release of H. axyridis adults. Flightless H. axyridis adults remained on experimental plants, in release plots, much longer than larvae [Both larvae and adults of a flightless strain of ltivars) . At an an larvae .H. axyridis were tested against A. solani on cultivated eggplants Solanum melongena (L.) [H. axyridis larvae per plant versus 2 adults per plant (60 plants per plot). Approximately 21 days after the third release of ladybird larvae and adults into separate test plots, 30 aphids per leaf  were on plants in the control (no-release) plots, and 10 aphids per leaf  were on plants in both test plots . Thus, A. solani population density was reduced by 66.7% in both test plots, when compared against the control plot  . A totalrol plot . Approxirol plot . Since tLife stage) using a comparative analysis of the results listed in r = 0.055; p = 0.81; n = 22); and ladybird larvae were less effective than adults, based on a ratio of aphid population reduction/release rate ; and larvae were not significantly less effective than adults  in suppressing aphids. Further research is necessary to confirm these results, especially because the number of studies involving releases of the flightless strain are limited in this review.In summarizing this section  larvae [The presence of other aphid predators on shared host plants could affect ladybird success, if ladybirds avoid these plants. If ladybirds forage on shared host plants, the interaction between predators could result in intraguild predation. Intraguild predation, in which one ladybird species attacks and kills another ladybird or non-ladybird predator, has been documented most often under laboratory conditions . For exaid flies ,62,63, a) larvae ,65. Howe) larvae .Coccinella transversoguttata Faldermann adults to reduce M. persicae densities in the presence or absence of two hemipteran predators, Geocoris bullatus (Say) and Nabis americoferus (Carayon), was tested on caged sugarbeet, Beta vulgaris vulgaris L. [C. transversoguttata adults reduced M. persicae populations when used alone or when combined with G. bullatus, N. americoferus, or G. bullatus adults. A release ratio of 4:114 (ladybird: aphid) resulted in a decline to less than 1 M. persicae per nine plants in six days. When 2 C. transversoguttata were combined with 4 G. bullatus or with 4 G. bullatus and 4 N. americoferus, it also resulted in a decline to less than 1 M. persicae in six days. Thus, aphid population reduction was 100% in all treatments (C. transversoguttata or any other predator species) contained an average of 416 M. persicae within the same time frame [When intraguild predation is limited, or non-existent, ladybirds can reduce aphid densities in greenhouses in the presence of other aphid predators. The ability of garis L. . C. traneatments . In contme frame .Undoubtedly, ladybirds and other aphid predators will occasionally come in contact with each other in greenhouses. Despite this fact, reliable quantitative data on the suppression of aphid populations under these conditions is scarce.H. axyridis and C. septempunctata larvae and adults) consumed un-parasitized aphids as readily as newly parasitized ones, but did not prefer consuming mummified aphids [The presence of aphid parasitoids on shared plants could affect the ability of ladybirds to reduce aphid densities. Aphid parasitoids and ladybirds typically interact when ladybirds come in contact with parasitized aphids. In the laboratory, ladybirds  and the parasitoid Aphidius colemani Viereck (adults at density of 1 adult/15 m2) were released in a glasshouse to suppress A. gossypii on strawberries [The frequency in which ladybirds prey on parasitized (and mummified) aphids in greenhouses is difficult to detect and quantify. The paucity of information on the net consequences of intraguild predation (of immature parasitoids) by ladybirds on aphid suppression in greenhouses signals a need for more research on this important topic. Nevertheless, the limited evidence suggests that the combined action of aphid parasitoids and ladybirds has a net positive effect on aphid population suppression (or limiting population growth) on plants. In a preliminary study, the convergent lady beetle wberries . Within wberries . HoweverH. axyridis complemented an aphid parasitoid, Aphelinus asychis Walker, in suppressing M. euphorbiae on cut roses Rosa hybrida L. in a replicate cage and open-release (without cages) trial [H. axyridis attacked M. euphorbiae and A. asychis mummies; H. axyridis larvae, not adults, showed a preference for killing and consuming aphids rather than mummies. In cages, M. euphorbiae peak densities were 75% lower when H. axyridis and mummies were present together, in comparison to cages with mummies alone. This cage study implies that H. axyridis complements the action of A. asychis despite the consumption of some of the parasitoid mummies. Note that H. axyridis adults escaped from the greenhouse after the first trial through a poorly fitting screen over the exhaust fan. After refitting the screen, a second release was more successful, with the establishment of adults which reproduced on plants. Soon after peak H. axyridis density was observed in the greenhouse, M. euphorbiae densities decreased more than 90% [H. axyridis was not released).The ladybird s) trial . Larva athan 90% . PerhapsC. septempunctata larvae and adults to suppress primarily A. gossypii and A. craccivora on sweet pepper (C. annuum) [A. colemani to suppress the aphid M. persicae in the same greenhouse. They found that A. colemani was capable of controlling low density populations of M. persicae. The authors did not indicate if C. septempunctata had a positive or negative effect on A. colemani. An ideal scenario would be the co-existence of ladybirds and aphid parasitoids with limited or no intraguild predation. The ability of aphid parasitoids to detect aphid aggregations in which a ladybird is foraging, or has been foraging, and to avoid those aggregations, has been revealed via laboratory and greenhouse cage experiments [Aphidius eadyi , Aphidius ervi , and Praon volucre , detected chemical trails on leaves visited by C. septempunctata and A. bipunctata [Researchers released  annuum) . In addieriments . Perhapseriments . Femalespunctata .In summarizing this section, ladybird predation of parasitized aphids (mummies) can occur on crop plants in greenhouses, but the net effect on aphid suppression is often positive or neutral, rather than negative. The actions of ladybirds and aphid parasitoids can increase the net negative effect on aphid population density. More research is necessary to convincingly demonstrate that the combination of ladybirds and parasitoids is more effective than either on their own for aphid suppression.Solenopsis invicta (Buren) reduced the survival of ladybirds (C. septempunctata and H. convergens larvae) by 50% on cotton G. hirsutum plants in cages, in comparison to controls (cotton plants in cages without fire ants) [The presence of ants on shared plants could affect the ability of ladybirds to reduce aphid densities. Antagonistic encounters between ants (Formicidae) and ladybirds have been reported most often on plants in open field conditions or in highly manipulated experiments in the laboratory. Foraging, predatory ants can negatively affect predation potential of ladybirds, when ants attack and kill larvae and deter adults from searching for aphids . For exare ants) .A. gossypii in caged tests and ladybird C. septempunctata and H. convergens larvae survival was reduced by 84% to 93% on cotton plants infested with A. gossypii in the presence of fire ants, as compared to when fire ants were absent [The mutualism between some ant and aphid species has been recognized for decades. Ants tend aphids for their honeydew and aggressively ward-off ladybirds, foraging for aphids . Thus, ae absent ,77.Scymnus species, to forage on aphid-infested plants and reduce aphid populations, even in the presence of tending ants, has been documented in field and laboratory studies [Scymnus larvae (and other Scymnini) could camouflage them from ants [Adalia, Coleomegilla, Harmonia, and Coccinella species) are not camouflaged from ant aggression; they do not have a wax covering. More research is necessary to document the chemical (molecular) basis of this camouflage hypothesis and to manipulate ant-aphid-ladybird densities in greenhouse experiments.Methods of subduing ant aggression against ladybird beetles are necessary to realize the full predation capacity of many ladybird species under pest management regimes in protected plant culture, when pesticides are not used. One possibility could involve using sticky barriers around the periphery and base of the crop plant ,79. An a studies ,81,82. Crom ants . Aphid-trom ants . It is pScymnus) seems to be undeterred by ants. Little or no research on the efficacy of Scymnus species under greenhouse conditions in the presence or absence of aphid-tending ants has been reported, to our knowledge. More research on this topic is necessary.To summarize this section, methods of subduing ant aggression against ladybird beetles on aphid infested plants are necessary to realize the full predation capacity of many ladybird species under organic management regimes in protected plant culture (greenhouses), without deploying pesticides. The predation capacity of some aphidophagous ladybird species  were more effective aphid predators in greenhouses.This review highlighted the effectiveness of several ladybird species as predators of aphids in greenhouses (and glasshouses). Aphid population reduction exceeded 50% in most studies and ladybird release rates usually did not correlate with aphid reduction. The ratio of aphid reduction/release rate was slightly less for larvae than for adults in some studies, suggesting that larvae were less effective (than adults) in suppressing aphids. Note that some of the adult releases were inside cages, thereby limiting adult dispersion from plants. Overall, the ratio of aphid reduction/release rate was greatest  for ladybird adults of several species of the normal strain, but least  for adults of a flightless Based on the limited number of species, and available studies, it was not clear which species was most effective. A comparison of predation capacity, voracity, body size, and tolerance to temperature extremes, amongst species, could help predict which species would be most effective in greenhouses. Many attack a range of aphid species and could be equally effective, if host plant defenses  do not disrupt ladybird foraging behavior. Integrating plant defenses with natural enemies to suppress arthropod pests provides challenges and opportunities .H. axyridis has been mass-produced and sold commercially for aphid control. Unfortunately, because of the rapid global expansion and negative effects that this species has purportedly had on other ladybirds (through intraguild predation), commercial production and sale of H. axyridis has waned. Although a flightless H. axyridis strain has been developed and proven effective for aphid control [From a practical standpoint, the choice of which species to use may heavily depend on the ease of rearing. For augmentative releases, mass rearing would be necessary to supply the large quantity of high-quality individuals required to reduce aphid densities . Note th control ,88, it m control .H. convergens adults to growers in North America. Adults are harvested from the Sierra Nevada (western USA) foothills each year [H. convergens adults have a strong flight propensity and tend to disperse from release sites within a few days [H. convergens adults, at extremely high release rates, did provide some level of aphid control in experimental nurseries [Because of the limited availability of mass-produced aphidophagous ladybirds currently on the market, a few biocontrol producers and retailers are involved in the acquisition and sale of field-collected, overwintered ach year . Many offew days . Their eurseries .Intraguild interactions between ladybirds and other predators, as well as aphid parasitoids, requires further study. Nevertheless, this review provides some evidence that intraguild predation does not deter ladybird predation of aphids. More research is necessary to determine if intraguild predation between ladybirds and generalist predators, such as minute pirate bugs, hoverflies, and lacewings , affects ladybird efficacy under greenhouse conditions. Interestingly, this review also provides evidence that ladybird predation of parasitoids (developing inside aphid prey) often does not hinder the suppression of aphid populations on plants.Scymnus species) that do not elicit ant aggression would be a worthwhile line of research.Encounters between ladybirds and foraging or aphid-tending ants in greenhouses has not been thoroughly studied. The few available studies clearly reveal that ants hinder most ladybirds, especially larvae, from attacking aphids. Physical barriers will be necessary to curb the entry of ants into greenhouses. Development of alternative methods to reduce ant aggression towards ladybirds on aphid-infested plants is also necessary. In addition, exploring the possibilities of mass producing and deploying ladybirds (e.g., some The conditions for successful aphid control by ladybirds in greenhouses has been reviewed. Despite the challenges, using ladybirds to manage aphid populations in greenhouses and similar protective structures is encouraged.