Huang et al. BMC Plant Biology (2015) 15:61 DOI 10.1186/s12870-015-0446-0 RESEARCH ARTICLE Open Access Structural, biochemical, and physiological characterization of photosynthesis in leaf-derived cup-shaped galls on Litsea acuminata Meng-Yuan Huang1†, Wen-Dar Huang2†, Hsueh-Mei Chou3, Chang-Chang Chen4, Pei-Ju Chen5, Yung-Ta Chang5* and Chi-Ming Yang6* Abstract Background: The source and sink relationships between insect-induced galls and host plant leaves are interesting. In this research, we collected cup-like galls induced by Bruggmanniella sp. (Diptera: Cecidomyiidae) on host leaves of Litsea acuminata and assessed them to investigate source-sink relationships between galls and host leaves. We characterized several of their photosynthetic characteristics including chlorophyll fluorescence (Fv/Fm), stomatal conductance, and photosynthetic capacity, biochemical components such as total soluble sugar, starches, free amino acids, and soluble proteins. The structural analyses were performed under confocal, light, and scanning electron microscopies. Results: Compared with host leaves, galls exhibited slightly lower chlorophyll fluorescence; however, stomatal conductance and photosynthetic capacity were not detected at all. Galls accumulated higher total soluble sugars and free amino acids but less soluble proteins than host leaves. No stomata was observed on exterior or interior gall surfaces under light or scanning electron microscopy, but their inner surfaces were covered with fungal hyphae. Confocal imagery showed a gradient of chloroplasts distribution between gall outer and inner surfaces. Conclusions: Our results strongly suggest that leaf-derived cecidomyiid galls are a type of chlorophyll-deficient non-leaf green tissue and consists on a novel sink in L. acuminate. Keywords: Cecidomyiidae, Gall, Litsea acuminate, Photosynthesis, Chlorophyll fluorescence, Sink Background specialized and nutritional relationships with their host Insect larvae residing inside galls use these leaf-derived plants because these insects spend major portions of structures as shelters for protection and sources of nu- their lives within galls. They interact with galls by the trition. More than 65% of galls, with various appearances simple removal of tissue or by damaging vascular tissues and colors, are derived from the leaves of their host in order to manipulate the synthesis and transport of plants within which the larvae reside. Three major hy- host plant nutrients [2-5]. Also, plants can use the galls potheses involving nutrition, environment, and enemies as sinks for nutrients for insects’ growth and reproduction have been postulated to explain the adaptive significance [6,7]. of gall induction and understand the evolution of gall We have previously pointed out that prior studies on morphology [1]. However, the source-sink relationships gall-caused impacts to host leaf photosynthesis do not between insect-induced galls and host leaves are still dis- suggest any general trends; however, Yang et al. [8,9] re- puted. Gall-inducing insects have developed highly ported a range of effects from negative to positive. This lack of pattern has not been confirmed within the past * Correspondence: [email protected]; [email protected] decade, therefore this question still remains under dis- †Equal contributors pute and requires further exploration. 5 Department of Life Science, National Taiwan Normal University, Taipei Regardless of whether net photosynthesis is directly 116Wenshan, Taiwan 6Biodiversity Research Center, Academia Sinica, Taipei 115Nankang, Taiwan measured in galls or estimated from radioactive labeling Full list of author information is available at the end of the article © 2015 Huang et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Huang et al. BMC Plant Biology (2015) 15:61 Page 2 of 12 experiments, the photosynthetic rates in galls are usually In previous studies, we concluded that two leaf- much lower than in unattacked normal leaf tissues [10]. derived cecidomyiid galls, the red ovoid galls induced by Aldea et al. [11] found lower photosystem (PS) II ef- D. taiwanensis and the green obovate galls induced by ficiency, as determined by chlorophyll fluorescence Daphnephila sueyenae, are photoassimilative sinks in (Fv/Fm), in Cecidomyia galls on Carya glabra leaves, Machilus thunbergii (Lauraceae) leaves. This data also Cynipid galls on Quercus velutina leaves, and erio- implies that insect-induced galls may have chlorophyll- phyid galls on Ulmus alata leaves compared to un- deficient non-leaf green tissues composed to a very high infected leaf surfaces. Photosynthetic rates of galled extent of heterotrophic tissues and autotrophic tissues leaves, measured by gas exchange, were reduced to a much lower extent [8,9,21]. when compared to ungalled leaves of naturally growing The ‘Ambrosia’ gall midges, significant portion of the Prunus serotina and Rhus glabra [12]. Water potential, family Cecidomyiidae, are one of the most diverse and photosynthesis rate (indicated by gas exchange), transpir- widespread groups of insects known to engage in symbi- ation, and stomatal conductance were decreased on leaves otic associations with fungi. The galls induced by these of Parthenium hysterophorus with Epiblema strenuana midges are typically lined internally with fungal hyphae, galls [13]. The Asian chestnut gall wasp was reported to which the developing larva may feed upon [22]. The reduce the photosynthesizing leaf area by around 40% cup-shaped gall induced by Bruggmanniella sp. also con- when compared to a non-galled leaf. It also induces tained an associated fungus. Litsea acuminata is an reductioninphotosyntheticcapacity(~60%)andsto- abundant and common subtropical tree species that is matal conductance (~50%) [14]. It has also been noted widely distributed in Taiwan. It is located 400 ~ 2,000 m that gall-inducing mites, such as Vasates aceriscrumena, may above sea level (asl) in Taiwan, and can grow to 20 m in be the major drivers of age-dependent reductions in the height with profuse branching. A cup-shaped gall in- physiological performance and growth of the canopy leaves duced by Bruggmanniella sp. on host leaves of L. acumi- of mature sugar maples (Acer saccharum) [15]. nata was examined to investigate the relationship In contrast, the phyllodes of Acacia pycnantha with between this gall and its host leaves [23]. Our field ob- wasp-induced galls had higher photosynthetic rates as servations revealed that Bruggmanniella larvae hatch indicated by gas exchange than similarly aged control from eggs in the spring, mine directly into leaf tissues, phyllodes without galls [10]. Photosynthesis (indicated and remain undeveloped until fall. Galls then begin to by gas exchange), stomatal conductance, and water po- develop around October and mature soon thereafter. tential were increased on Silphium integrifolium leaves The larvae develop into second and third instars within with Antistrophus silphii galls compared to ungalled mature galls and emerge in early spring of the following shoots [16]. A scale insect on leaves of Ilex aquifolium year. also caused a higher PSΙΙ energy transduction efficiency, Little study has been done on the photosynthetic char- as indicated by chlorophyll fluorescence (Fv/Fm), in af- acteristics of gall midges and their relationship to the fected tissues relative to uninfected tissues [17]. photosynthetic biochemical mechanisms of galls. In this The characterization of gall transcriptomes in grape study, we investigated the effects of galling by a midge leaves shows that galling insects increase their primary on L. acuminata by measuring chlorophyll fluorescence, metabolic gene expression, including glycolysis, fer- photosynthetic capacity, ultrastructural morphology, and mentation, and the transport of water, nutrients, and biochemical composition of the gall and the host leaf. minerals in leaf-derived gall tissues, and decrease the expression of genes responsible for non-mevalonate and terpenoid synthesis, but increase the biosynthesis Results of shikimate and phenylpropanoid, which are secondary Photosynthetic pigments metabolites that alter the defense status of grapes [18]. Host plant leaves and their galls have different Car/Chl Investigation of the metabolic responses of pteromalid ratios in addition to great differences in Chl and Car wasp (Trichilogaster acaciaelongifoliae)larvaeinbud content (Table 1). While galled or gall-free leaves con- galls on Acacia longifolia to reduced oxygen (O2)and tained around 2,000 and 1,000 μg/g DW of Chl and Car, elevated carbon dioxide (CO2) indicates that the larvae respectively, and gall levels were reduced to 39 and are tolerant to hypoxia/hypercarbia and are capable of 21 μg/g DW, respectively. That is, both the Chl and Car reducing their respiratory rates to cope with hypercar- content of galls are only ~2% of the gall-free or galled bia [19]. Symbiosis between gall-inducing insects and leaves. While all the Chl a/b ratios of galls, and galled fungi catalyze their expansion of resource use (niche and gall-free leaves were the