Olivier Gnankine ´

Laurence Mouton

N E Henri

Gabriel Terraz

Thomas Houndete

Thibaud Martin

Fabrice Vavre

Fre ´de

´ric Fleury

Olivier Gnankine´
email: olgnankine@hotmail.com

Distribution of Bemisia tabaci (Homoptera: Aleyrodidae) biotypes and their associated symbiotic bacteria on host plants in West Africa

Keywords: Bemisia tabaci, biotypes, host plant, secondary symbionts, West Africa

.

2. Our study describes the biotypes and symbiotic bacterial communities of B. tabaci sampled in three West African countries, Burkina Faso, Benin, and Togo. A total of 527 individuals were collected from seven cultivated host plants.

3. In the 20 localities studied, we found the same three biotypes AnSL, ASL, and Q previously detected in Burkina Faso. These biotypes display a specific pattern of geographical distribution influenced by the host plant species. In Benin and Togo, the ASL and AnSL biotypes were predominant, while in Burkina Faso, the Q biotype was dominant, with two sub-groups, Q1 and Q3 (recorded to date only in this country), and ASL individuals found in sympatry with Q1 individuals in some localities. As previously reported, each biotype and each genetic group harbours a specific community of symbiotic bacteria.

Introduction

The whitefly Bemisia tabaci (Gennadius) is a significant pest that damages many agricultural crops worldwide. B. tabaci is in fact a complex of cryptic species composed of 11 clearly differentiated, high-level groups containing at least 24 distinct species composed of several low-level groups previously designated as 'biotypes' [START_REF] De Barro | Bemisia tabaci: a statement of species status[END_REF][START_REF] Dinsdale | Refined global analysis of Bemisia tabaci (Hemiptera: Sternorrhyncha: Aleyrodoidae: Aleyrodidae) mitochondrial cytochrome oxidase 1 to identify species level genetic boundaries[END_REF]. Many aspects of this species complex remain unknown, such as the origins of the different species and the process of radiation, the borders between the species and their degree of genetic isolation, the geographical distribution, the biological characteristics and the within-species genetic diversity. B. tabaci biotypes can be distinguished using biochemical or molecular markers (allozymes, mitochondrial Cytochrome Oxidase CO1, nuclear Internal Transcribed Spacer ITS), but their biological significance and taxonomic status are still unclear. These biotypes can differ with regard to the induction of plant silverleafing disorders, host range, fecundity, insecticide resistance, ability to transmit plant viruses [START_REF] Perring | The Bemisia tabaci species complex[END_REF][START_REF] De Barro | Bemisia tabaci: a statement of species status[END_REF], and in the symbiotic bacterial community they harbour [START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF]. The situation is in fact even more complex, as significant genetic differentiation is also observed within biotypes. For example, within the Q biotype of the Mediterranean species, the genetic sub-groups designated Q1, Q2, and Q3 have been recognised both by CO1 sequencing and from the composition of their endosymbiotic bacteria [START_REF] Mckenzie | Distribution of Bemisia tabaci (Hemiptera: Aleyrodidae) biotypes in Florida-investigating the Q invasion[END_REF][START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF].

In tropical and sub-tropical countries, B. tabaci is a pest of primary importance, especially on crops of cassava, cotton, sweet potatoes, tobacco, and tomato. In West Africa, population outbreaks were observed in 1998 in cotton fields in Burkina Faso, Mali, and Ivory Coast inducing severe crop damages [START_REF] Otoidobiga | Susceptibility of fiel populations of adult Bemisia tabaci (Homoptera: Aleyrodidae) and Eretmocerus sp. (Hymenoptera: Aphelinidae) on the population dynamics of the pest in Burkina Faso (West Africa)[END_REF][START_REF] Otoidobiga | Field efficacy and baseline toxicities of pyroproxifen, acetamiprid and diafenthiuron Bemisia tabaci Gennadius (Homoptera: Aleyrodidae) in Burkina Faso (West Africa)[END_REF]. The resulting losses had a severe impact on the economic activity of these countries, as cotton production and trading is one of the main financial resources, especially in Burkina Faso. As a consequence, West African countries make wide use of many pesticides to control B. tabaci populations despite their harmful impact on potential natural enemies and on the environment. This practice also leads to high levels of resistance, which have been observed particularly in Burkina Faso [START_REF] Hounde´te´ | Insecticide resistance in field populations of Bemisia tabaci (Hemiptera: Aleyrodidae) in West Africa[END_REF]. As B. tabaci biotypes have differing levels of insecticide resistance [START_REF] Horowitz | Biotypes B and Q of Bemisia tabaci and their relevance to neonicotinoid and pyriproxyfen resistance[END_REF][START_REF] Wang | Biotype and insecticide resistance status of the whitefly Bemisia tabaci from China[END_REF] and do not always share the same resistance mutation to a particular chemical class of insecticide [START_REF] Alon | Multiple origins of pyrethroid resistance in sympatric biotypes of Bemisia tabaci (Hemiptera: Aleyrodidae)[END_REF][START_REF] Alon | Organophosphates' resistance in the B-biotype of Bemisia tabaci (Hemiptera: Aleyrodidae) is associated with a point mutation in an ace1type acetylcholinesterase and overexpression of carboxylesterase[END_REF], pest management programs may inadvertently select for one of these biotypes locally. For instance, the polyphagous B biotype is known to be more susceptible to several chemical compounds than the Q biotype [START_REF] Horowitz | Biotypes B and Q of Bemisia tabaci and their relevance to neonicotinoid and pyriproxyfen resistance[END_REF], which explains why the Q biotype sometime displaces the B biotype [START_REF] Horowitz | Biotypes B and Q of Bemisia tabaci and their relevance to neonicotinoid and pyriproxyfen resistance[END_REF][START_REF] Chu | Change in the biotype composition of Bemisia tabaci in Shandong Province of China from 2005 to 2008[END_REF]but see Crowder et al., 2010). Another consequence is that the effectiveness of treatments may vary depending on the local composition of biotypes and the chemical classes of insecticides used. Identification of the biotypes present and information about their diversity are thus clearly required for effective control strategies of this pest.

Although major studies on B. tabaci have been performed in East Africa, little is known about the distribution and frequency of the various biotypes in West Africa. So far, three biotypes have been reported in this region: the Sub-Saharan African non-Silver Leafing (AnSL) biotype, the Sub-Saharan African Silverleafing (ASL) biotype, and more recently, the Q biotypes [START_REF] Berry | Molecular evidence for five distinct Bemisia tabaci (Homoptera: Aleyrodidae) geographic haplotypes associated with cassava plants in sub-Saharan Africa[END_REF][START_REF] Brown | Genetic differentiation of whitefly Bemisia tabaci mitochondrial cytochrome oxidase I, and phylogeographic concordance with the coat protein of the plant virus genus Begomovirus[END_REF][START_REF] Sseruwagi | Genetic diversity of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) populations and presence of the B biotype and a non-B biotype that can induce silverleaf symptoms in squash, in Uganda[END_REF][START_REF] De La Rua | New insights into the mitochondrial phylogeny of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) in the Mediterranean Basin[END_REF][START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF]. The AnSL biotype belongs to the Sub-Saharan African clade, and is considered to be the basal group of the species complex, and probably originated in Africa. AnSL has been reported throughout Africa, except in the northern countries near the Mediterranean basin [START_REF] Berry | Molecular evidence for five distinct Bemisia tabaci (Homoptera: Aleyrodidae) geographic haplotypes associated with cassava plants in sub-Saharan Africa[END_REF][START_REF] Boykin | Global relationships of Bemisia tabaci (Hemiptera: Aleyrodidae) revealed using Bayesian analysis of mitochondrial COI DNA sequences[END_REF][START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF]. The closely related ASL and Q biotypes are genetically distant from AnSL. They both belong to the Mediterranean species within the Africa ⁄ Middle East ⁄ Asia Minor group [START_REF] Boykin | Global relationships of Bemisia tabaci (Hemiptera: Aleyrodidae) revealed using Bayesian analysis of mitochondrial COI DNA sequences[END_REF][START_REF] De Barro | Bemisia tabaci: a statement of species status[END_REF]. ASL has been reported in Ghana, Ivory Coast, Nigeria, Benin, Togo, and Burkina Faso [START_REF] Brown | Genetic differentiation of whitefly Bemisia tabaci mitochondrial cytochrome oxidase I, and phylogeographic concordance with the coat protein of the plant virus genus Begomovirus[END_REF][START_REF] De La Rua | New insights into the mitochondrial phylogeny of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) in the Mediterranean Basin[END_REF][START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF]. Recently, we reported that the Q biotype was present in Burkina Faso, where we detected two (Q1 and Q3) of the three genetic subgroups previously described [START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF]. However, there is a paucity of data for West Africa, and the precise geographical distribution, the frequency of the biotypes, and their genetic subdivisions, together with their possible association with symbiotic bacteria and host plant species remain largely unknown.

The present study set out to describe the distribution of the B. tabaci biotypes across West African countries, and its relationship with the host plant species. A total of 527 individuals collected from 20 localities in three countries (Burkina Faso, Benin and Togo), and from seven host plant species, were sampled during the rainy season in 2007 and 2009. In addition to identifying the biotype and genetic group, we determined the composition of the symbiotic bacteria community they harboured. Indeed, B. tabaci, like most phloem-feeding insects, is host to an obligatory primary endosymbiont, the bacterium Portiera aleyrodidarum, which is required to provide essential nutrients for its host's survival and development. B. tabaci is also infected by several facultative, vertically transmitted, symbiotic bacteria known as secondary endosymbionts [START_REF] Zchori-Fein | Diversity of prokaryotes associated with Bemisia tabaci (Gennadius) (HemipteraAleyrodidae)[END_REF]. Interestingly, each biotype or genetic group is infected by a specific symbiotic community [START_REF] Chiel | Biotype-dependent secondary symbiont communities in sympatric populations of Bemisia tabaci[END_REF][START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF]. This raises a question about the role of these symbiotic microorganisms in the biology of B. tabaci, and in the process of species radiation. Their phenotypic effects remain largely unknown in B. tabaci, but in other insect species, bacteria are known to be able to manipulate host reproduction [START_REF] Stouthamer | Wolbachia pipientis: Microbial manipulator of Arthropod reproduction[END_REF][START_REF] Weeks | Incidence of a new sex-ratio-distorting endosymbiotic bacterium among arthropods[END_REF], and to be involved in resistance to natural enemies [START_REF] Oliver | Facultative bacterial symbionts in aphids confer resistance to parasitic wasps[END_REF] or host-plant specialisation [START_REF] Tsuchida | Host plant specialization governed by facultative symbiont[END_REF]. In B. tabaci, Rickettsia bacteria enhance susceptibility to insecticides [START_REF] Kontsedalov | The presence of Rickettsia is associated with increased susceptibility of Bemisia tabaci (Homoptera: Aleyrodidae) to insecticides[END_REF], but can also enhance whiteflies performance [START_REF] Himler | Rapid spread of a bacterial symbiont in an invasive whitefly is driven by fitness benefits and female bias[END_REF]. Moreover, symbiotic bacteria are thought to play a major role in the virus transmission capacities of the whitefly [START_REF] Morin | A GroEL homologue from endosymbiotic bacteria of the whitefly Bemisia tabaci is implicated in the circulative transmission of tomato yellow leaf curl virus[END_REF][START_REF] Gottlieb | The transmission efficiency of tomato yellow leaf curl virus by the whitefly Bemisia tabaci is correlated with the presence of a specific symbiotic bacterium species[END_REF].

The main objective of this study was to provide the first detailed description of the diversity of B. tabaci biotypes and their symbiotic bacteria communities in West Africa on several host plant species. This provides a good basis for answering questions about biotype dynamics in relation to insecticide resistance, as well as providing valuable information for developing integrated pest management programmes.

Materials and methods

Origin of the whiteflies

Sampling was performed in 2007 and 2009. Live adult B. tabaci (males and females) were collected and individually placed in 1.5-ml tubes containing 95% ethanol and kept at )20°C until they underwent DNA extraction. The samples came from three neighbouring countries in West Africa: Burkina Faso, Benin, and Togo (Fig. 1). A total of 527 individuals were collected at 20 localities and from seven cultivated host plants (cotton, tomato, okra, tobacco, marrow, Lantana camara, cassava) (Table 1). At least eight individuals were analysed per locality and per plant. The cotton, tomato, marrow, and okra plantations had all been treated with insecticides (with the exception of the cotton from Kandi Sina in Benin) belonging to several classes: organophosphates, pyrethroids, and neonicotinoids. No treatment had been applied to the cassava and Lantana camara (Table 1).

DNA extraction

Total DNA was extracted from each individual in 26 ll of an extraction buffer containing 50 mM KCl, 10 mM Tris-base pH 8, 0.45% Nonidet P-40, 0.45% Tween 20 and 50 mg ml )1 Proteinase K. Extraction buffer was added to the crude extract, incubated at 65°C for 3h and then incubated at 100°C for 15 min. A volume of 35 ll of pure water was added to this extract, which was then stored at )20°C until use.

Identification of B. tabaci biotypes and genetic subgroup

The biotypes were identified on the basis of the mitochondrial marker gene COI (Cytochrome Oxidase 1) after a PCR reaction using the universal COI primers C1-J-2195 (5¢-TTGATTT TTTGGTCATCCAGAAGT-3)' and L2-N-3014 (5¢-TCCAA TGCACTAATCTGCCATATTA-3¢) [START_REF] Khasdan | DNA markers for identifying biotypes B and Q of Bemisia tabaci (Hemiptera: Aleyrodidae) and studying population dynamics[END_REF]. PCR reactions were performed in 25-ll volumes containing 200 lM dNTP, 200 nM primers, 0.5 IU Taq DNA polymerase (Eurobio) and 2 ll of DNA template. Cycling conditions consisted of an initial denaturing step at 95°C for 5 min, followed by 30 cycles of 1 min at 95°C for denaturing, 1 min at 50°C for annealing, 1 min at 72°C for extension, and a final extension of 72°C for 10 min (TetradÒ, Biorad, France). For some individuals (from 2 to 20 individuals per population), the PCR products were then sequenced to determine the biotype. For the others, we used the PCR-RFLP (PCR-Random Fragment Length Polymorphism) test developed and described by H. Henri, G. Terraz, O. Gnankine´, F. Fleury & L. Mouton (unpubl. data). Briefly, digestions of the mtCOI amplifications by the XapI and BfmI restriction enzymes result in different patterns of fragment migration depending on the B. tabaci genetic group. Products are visualised in 2% agarose gels containing ethidium bromide (0.5 lg ml )1 ) under UV illumination after a 1-h migration at 100 mV. This PCR-RFLP test can be used to identify all known genetic groups from Africa ⁄ Middle East ⁄ Asia Minor, and major clades, including the Q1, Q2, and Q3 groups of the Mediterranean Q biotype.

Detection and molecular identification of the secondary endosymbiotic bacteria (SS)

Individuals were screened for symbiotic bacterial infection using specific PCR primers that amplify the 16S rRNA gene for Hamiltonella, Cardinium, and Rickettsia, the 23S rRNA gene for Arsenophonus and Fritschea and the wsp gene for Wolbachia (Table 2). The presence of the obligatory symbiont Portiera was also checked to confirm the quality of DNA extraction. DNA was amplified in a final volume of 25 ll, containing 1.5 mM MgCl2, 200 lM dNTPs, 200 nM of each primer, 0.5 IU Taq DNA polymerase (Eurobio) and 2 ll of DNA. PCR products were visualised in 1% agarose gels stained with ethidium bromide (0.5 lg ml )1 ) under UV illumination to check for the presence ⁄ absence of the various bacteria.

Phylogenetic analyses

Phylogenetic analyses of B. tabaci biotypes were performed on mitochondrial COI sequences. The accession numbers of the sequences obtained in the present study and used to construct the tree are listed in Table 3. Sequences of B. tabaci from Genbank were also included (Fig. 2). Multiple sequence alignment was carried out using MUSCLE software [START_REF] Edgar | MUSCLE: multiple sequence alignment with hugh accuracy and high throughput[END_REF]. The appropriate model of evolution was estimated with jModeltest v 0.1.1 [START_REF] Posada | JmodelTest: phylogenetic model averaging[END_REF]. The best likelihood score was evaluated with a corrected version of the Akaike information criterion for small samples (AICc). The model selected was HKY+G. Using this model, the phylogenetic tree was constructed by the maximum likelihood method with PhyML (version 3.0.1) [START_REF] Guindon | A simple, fast and accurate algorithm to estimate large phylogenies by maximum likelihood[END_REF] and edited using Treedyn [START_REF] Chevenet | TreeDyn: towards dynamic graphics and annotations for analyses of trees[END_REF]. The robustness of the each node was assessed from 100 bootstrap replicates.

Statistical analyses

The data collected were subjected to Fisher's exact test with simulated p-values based on 2000 replicates using R statistical software (http://www.r-project.org/).

Results

Geographical distribution of biotypes

Among the 527 individuals sampled from 20 localities in Burkina Faso, Togo, and Benin, three genetic groups recognised as biotypes were identified (Table 1): Q, ASL and the basal group AnSL. The findings revealed a clear distinction between the different countries (Fig. 1). In Togo and Benin, the African AnSL and the ASL biotypes of B. tabaci were predominant with two clearly differentiated genetic groups within the AnSL (2004). These two sub-groups were found on cassava only and occurred together in sympatry (Table 1). However, ASL was the dominant biotype in this area (100% at 8 out of 11 sites). In 2009, the Q biotype was also recorded, but only in the northern parts of these countries. In Tove´(Togo) and Kandi (Benin), this biotype was always observed in sympatry with ASL and at a low frequency, whereas at Dapaong (Togo), all individuals belonged to this biotype (Table 1; Fig. 1). The situation was quite different in Burkina Faso, where the Q biotype dominated at most of the sites sampled. The ASL biotype occurred at low frequency and always in sympatry with Q1 (Table 1; Fig. 1), and the AnSL biotype was never detected. However, it should be pointed out that no sample was available from cassava. Sequence analyses revealed the presence of two genetic sub-groups of the Q biotype: Q1 and the newly described Q3 sub-group recently detected in West Africa [START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF]. Q1 and Q3 showed different distribution patterns in Burkina Faso in terms both of locality and host plant species. Q3 was only observed in the Western part of the country, and only on L. camara and tobacco, whereas Q1 was found at most sampling sites and on most host plants (Fig. 2). Finally, ASL was the only biotype recovered from all three countries, albeit at different frequencies.

Distribution of biotypes on host plants

As the samples were not always taken from the same host plants in all three countries, the plants sampled and the locality were not independent. This made it impossible to carry out a global statistical test to find out whether the host plant influences the distribution of the biotypes. However, it did look as though there was a strict association between the AnSL biotype and cassava, no other biotype having ever been found on this plant, even when ASL was present in the same locality (Table 1). AnSL was never found on cotton crops, regardless of whether they had undergone insecticide treatment or not. Similarly, only the Q3 group was found on L. camara. Although Q3 individuals were also observed on tobacco, it was only on L. camara that this biotype was not found in sympatry with other biotypes. Importantly, these two plants were the only ones that had not been treated with insecticides in our Burkina Faso samples, raising the possibility that the distribution of Q3 is mainly determined by insecticide use. In Bobo ⁄ Kuinima (Burkina Faso), where four plant species had been sampled in 2007, a significant relationship was found between the plant and the biotype distribution (Fisher's Exact Test, P = 0.0005). In particular, Q3 was only found on tobacco, and was strongly predominant on this plant. Observation of biotype distribution among the host plants also suggests an association between the Q1 biotype and cotton, since when it is present Q1 reaches very high frequencies of up to 100% on this plant. Once again, it is possible that this pattern was mediated by insecticide use.

Prevalence, diversity, and distribution of endosymbiotic bacteria

The presence of the seven known symbiotic bacteria infecting B. tabaci primary symbiont Portiera and the six secondary symbionts: Hamiltonella, Arsenophonus, Rickettsia, Cardinium, Wolbachia, Fritschea) was checked by specific diagnostic PCR. As expected, all individuals were found to be infected with Portiera, which demonstrated the high quality of the DNA extractions. Figure 3 shows the infection status of individuals from all 20 localities and from different host plants with regard to all the secondary endosymbionts except Fritschea, which was never detected. As previously reported [START_REF] Chiel | Biotype-dependent secondary symbiont communities in sympatric populations of Bemisia tabaci[END_REF][START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF], all genetic groups (AnSL1, AnSL2, ASL, Q1, Q3) were found to host a specific bacterial assemblage (Fisher's Exact Test, P = 0.0005): the most common bacteria being Hamiltonella for the Q1 biotype, Arsenophonus for ASL and AnSL biotypes, and the association Arsenophonus ⁄ Rickettsia for the Q3 group. However, the infection status of individuals belonging to the ASL biotype is variable, with a strong contrast being observed between Burkina Faso and Togo ⁄ Benin (Fisher's Exact Test, P = 5 • 10 )15 ). Indeed, while in Burkina Faso most of the individuals are infected by Arsenophonus (80% on average), the level of infection with this secondary symbiont was low in Togo and Benin (less than 12%), and it was not detected at all on some populations in Benin (Fig. 3). Here, we report for the first time the infection status of the basal clade AnSL, which appears to be very similar to that of the ASL biotype, having Arsenophonus as the main symbiotic bacterium and a high frequency of uninfected individuals.

In contrast to the findings of a previous study [START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF], multiple infections with several secondary symbionts were uncommon in the areas we sampled (13.7%; Fig. 3). In most cases, they involved infection with two species (95.8%) and were mainly observed within the Q3 biotype co-infected by Rickettsia and Arsenophonus.

Discussion

This first detailed analysis of B. tabaci biotypes in West African countries provides valuable information about the level of regional biodiversity, including cytoplasmic biodiversity due to infection by symbiotic bacteria related to different host plant species and insecticide treatments. It also extends our knowledge about the geographical range of major biotypes that have spread throughout the world, such as the Q biotype. We identified the same biotypes as had previously been reported in Burkina Faso by [START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF], i.e. the ancestral AnSL (Sub-Saharan Africa non-Silver Leafing), the ASL (SubSaha-ran Africa Silver Leafing) and the Q biotypes. The frequencies of these biotypes showed marked differences depending on the site sampled. One central finding is that the Q biotype was present at a high frequency in Burkina Faso in 2007 and 2009, whereas it was only detected in the northern parts of Togo and Benin in 2009, which could be explained by a recent introduction into these latter countries of agricultural practices that facilitate its ability to spread. In addition to biotype diversity, the COI mitochondrial marker revealed the existence of subgroups within the AnSL and Q biotypes (designated AnSL1, AnSL2 and Q1, Q3 respectively). The detection of diversity at such a small geographical scale raises numerous questions about the evolution of biotypes, the coexistence and distribution of sub-groups, and the consequences of this diversity for B. tabaci management.

These three biotypes (ASL, AnSL, and Q) have also been detected in East Africa, including five sub-groups of the AnSL biotype, but only one, the Q1 sub-group, of biotype Q [START_REF] Berry | Molecular evidence for five distinct Bemisia tabaci (Homoptera: Aleyrodidae) geographic haplotypes associated with cassava plants in sub-Saharan Africa[END_REF][START_REF] Brown | Genetic differentiation of whitefly Bemisia tabaci mitochondrial cytochrome oxidase I, and phylogeographic concordance with the coat protein of the plant virus genus Begomovirus[END_REF][START_REF] Sseruwagi | Genetic diversity of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) populations and presence of the B biotype and a non-B biotype that can induce silverleaf symptoms in squash, in Uganda[END_REF][START_REF] De La Rua | New insights into the mitochondrial phylogeny of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) in the Mediterranean Basin[END_REF][START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF]. Sub-group Q3 had only been detected in Burkina Faso, but this could have been due to a sampling bias, as Q3 appears to be restricted to certain plants (tobacco and L. camara). In both East and West Africa, the Q biotype has been found at higher latitudes than the ASL and AnSL biotypes. In northern African countries, such as Morocco and Tunisia, the Q1 biotype is predominant [START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF], and a similar trend can also be seen in the present study (Fig. 1). In East Africa, the Q biotype has only been detected in Sudan, which is in the North ⁄ Centre region of Africa, and in the Uganda, which borders it to the south. In countries further south (Tanzania, South Africa, Mozambique), AnSL is widespread. However, Cameroon, Benin, and Togo, where we almost exclusively find the AnSL biotype, are at a higher latitude than Uganda, so latitude alone cannot entirely explain the pattern of distribution, and longitude must also be taken into account.

Interestingly, in Burkina Faso, ASL was always found in sympatry with the Q biotype and on the same host plant, particularly the Q1 sub-group, suggesting the possible coexistence of the ASL and Q biotypes at least on a short time scale. However, on all these plants, Q1 was predominant. On cotton, ASL was rarely observed and when present, it never reached frequencies of more than 7%. This situation could indicate competitive interactions between ASL and Q in Burkina Faso, with the outcome of competition depending on environmental conditions, such as host plant species or agricultural practice. Indeed, resistance to neonicotinoids has been shown in this country to be correlated with the presence of the Q1 biotype, with ASL individuals being most susceptible (O. Gnankine´, L. Mouton, A. Savadogo, T. Martin, A. Sanon, F. Vavre, R. Dabire & F. Fleury, unpubl. data). More generally, the Q biotype is known to be highly resistant to several insecticides [START_REF] Horowitz | Biotypes B and Q of Bemisia tabaci and their relevance to neonicotinoid and pyriproxyfen resistance[END_REF], which could give it a competitive advantage when insecticides are used. This situation had already been observed among the B and Q biotypes, the latter excluding the former in the context of insecticide treatment [START_REF] Chu | Change in the biotype composition of Bemisia tabaci in Shandong Province of China from 2005 to 2008[END_REF][START_REF] Crowder | Mating behaviour, life history and adaptation to insecticides determine species exclusion between whiteflies[END_REF]. The same phenomenon may occur in Western Africa among resistant Q and susceptible ASL biotypes, explaining Distribution of Bemisia tabaci biotypes and their associated symbiotic 417 Ó 2012 The Authors Insect Conservation and Diversity Ó 2012 The Royal Entomological Society, Insect Conservation and Diversity, 6, 411-421 why the sensitivity of adult populations of B. tabaci can vary greatly from one place and 1 year to another in Burkina Faso [START_REF] Gnankine´ | Traitements insecticides et dynamique des populations de Bemisia tabaci en culture cotonnie`re au Burkina Faso[END_REF]. However, in the localities where we sampled twice, in 2007 and in 2009, the prevalence of these biotypes was similar. This time lapse may have been too short, and other records on the spatial distribution of biotypes in Burkina Faso and in bordering countries spanning several are required before we can determine whether the Q biotype is actually spreading, and whether the sympatric situation of ASL and Q is stable or whether one biotype will eventually exclude the other, which could have major repercussions for pest management and agricultural production in this region.

The stable co-existence of biotypes could involve host-plant specialisation; for example, AnSL was the only biotype found on cassava. An earlier survey in sub-Saharan Africa [START_REF] Abdullahi | Molecular characterization of whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae) populations infesting cassava[END_REF] had already highlighted the fact that the populations that develop on cassava seem to be restricted to cassava only, whereas populations from other plants are polyphagous, but do not colonise cassava. Such niche specialisation of the AnSL, with cassava exercising a repulsive action on other biotypes, could allow it to persist even if other biotypes are present locally. Unfortunately, all our samples collected on cassava originated from Togo and Benin, where ASL was the predominant biotype. Sampling on cassava in Burkina Faso could reveal whether AnSL is also found in this country despite the presence of Q1 and Q3. Q3 also seems to develop specifically in one host plant, L. camara, where no other biotype has been detected, which suggests that even within the Q biotypes, there can be significant ecological and biological differences between sub-groups. However, this effect could also be due to the fact that this plant did not undergo insecticide treatment, something also suggested by the presence of Q3 on untreated tobacco. Further investigations of the ability of Q1, ASL and Q3 to develop on these different plants and on their resistance to insecticides are required to clarify and allow up to predict the dynamics of these biotypes and sub-groups.

) 0% H C W R A 0% H C W R A 100% 0% H C W R ASL Q1 A (BF-Cott4a) Q1, ASL (n = 21) Diabo -Cotton (BF-Cott4b) Q1, ASL (n = 20) H C W R ASL Q1 A 100% 0% 100% 0% H C W R ASL Q1 A Bobo/Kuinima-Tomato (BF-Tom5a) Q1, ASL (n = 33) H C W R ASL Q1 A 100% 0% Bobo/Kuinima-Marrow (BF-Mar5a) Q1, ASL (n = 31) H W R A Q1 Q3 ASL Bobo/Kuinima-Tobacco (BF-Tob5a) Q1, Q3, ASL (n = 30) 100% 0% H C W R A ASL Q1 Bobo/Kuinima-Okra (BF-Okr5a) Q1, ASL (n = 15) 100% 0% 100% 0% H C W R ASL Q1 A 100% 0% H C W R ASL Q1 A Ouaga/Barrage3-Tomato (BF-Tom7a) Q1, ASL (n = 15) 100% 100% 0% H C W R A (BF-Cam7a) Q3 (n = 44) Univ-Ouaga1-L. camara (BF-Cam7b) Q3 (n = 20) 0% H C W R A 100% Bobo/Farako-Bâ-Cotton (BF-Cott6a) Q1, ASL (n = 15) (
Benin 100% 0% H C W R A Tanwé-Cotton (Be-Cott1a) ASL (n = 10) 100% 0% H C W R A Koui-Cotton (Be-Cott2a) ASL (n = 18) 100% 0% H C W R A 100% 0% H C W R A IITA-Tomato (Be-Tom4a) ASL (n = 10) 100% 0% H C W R A (Be-Cott5a) ASL (n = 10) CRA-CF/Bohicon-Cotton (Be-Cott5b) ASL (n = 11) 0% H C W R A 100% 100% 0% H C W R A Sémé-Tomato (Be-Tom6a) ASL (n = 10) 100% 0% H C W R AnSL 1 2 A Agonkanmey-Cassava (Be-Cas7a) AnSL (n = 16) Kandi Sina-Cotton (Be-Cott8b) Q1, ASL (n = 21) Togo 100% 0% H C W R A H C W R
Our study also confirms the initial observations that there is a link between symbiotic bacterial communities and the biotypes or genetic groups of B. tabaci [START_REF] Chiel | Biotype-dependent secondary symbiont communities in sympatric populations of Bemisia tabaci[END_REF][START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF]. As previously observed, within the Q biotype, most Q1 individuals harbour Hamiltonella and sometimes low frequencies of Wolbachia or ⁄ and Cardinium, whereas Q3 individuals were mostly found to harbour Arsenophonus with a high level of co-infection with Rickettsia. In the ASL and AnSL biotypes, Arsenophonus is the most common bacterium with higher frequency in the AnSL than the ASL biotype. In very few cases, other bacteria were reported such as Wolbachia, Rickettsia, or Hamiltonella. The reasons for these biotype-symbiont specific associations remain unknown, and the first step towards understanding the mechanisms involved will be to determine the phenotypic effects of these bacteria in B. tabaci. In aphids, some secondary symbionts are known to be involved in ecological specialisation, such as host plant utilisation [START_REF] Tsuchida | Host plant specialization governed by facultative symbiont[END_REF], and so the symbiotic community could play a major role in biotype adaptation and competition.

One noteworthy finding is the fact that the prevalence of secondary symbionts varied considerably between the different localities and genetic groups. Most individuals from Burkina Faso belonged to the ASL and Q biotypes, and displayed high levels of infection, while in Benin and Togo, where the Q biotype is rare, the prevalence of secondary symbionts in ASL, consisting mainly Arsenophonus, was very low. Moreover, in Benin, some populations did not harbour any of the 6 secondary symbionts we checked. This is the first description of B. tabaci populations in which no secondary symbionts have been recorded. In addition, only a small number of individuals were infected by more than one secondary symbiont, with the exception of individuals belonging to the Q3 biotype, which contrasts with a previous study in which a high level of multiple infections was found [START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF]. However, it is impossible to rule out the possibility that other bacterial symbionts were in fact present in some B. tabaci individuals, and further studies involving more detailed investigations of the symbiotic content are called for. However, despite these limitations, the data reported raise several questions about the dynamics of infection by secondary symbionts and the influence of the environment on symbiotic composition.

  Distribution of Bemisia tabaci biotypes and their associated symbiotic 413 Ó 2012 The Authors Insect Conservation and Diversity Ó 2012 The Royal Entomological Society, Insect Conservation and Diversity, 6, 411-421 biotype, designated AnSL1 and AnSL2 (Fig. 2), which correspond to the Sub Saharan Africa 1 and 2 genetic groups, respectively, described in Dinsdale et al. (2010) and Berry et al.

Fig. 1 .

 1 Fig. 1. Geographical distributions of the samplings sites and of Bemisia tabaci biotypes.

Fig. 2 .

 2 Fig. 2. COI-gene-based phylogeny of Bemisia tabaci.

Fig. 3 .

 3 Fig. 3. Infection status of Bemisia tabaci individuals collected in 16 localities of three Western Africa countries.

  Fig. 3. (Continued).

Table 1 .

 1 Frequencies of Bemisia tabaci biotypes related to host plants and localities.

					Agricultural		Year
	Country	Acronym*	Locality	Host plant	practice	nà	collected	Biotypes present (%) §
	Burkina	BF-Cott1a	Kompienga	Cotton	Treated	15	2007	Q1 (100)
	Faso	BF-Cott1b		Cotton	Treated	21	2009	Q1 (96), ASL (4)
		BF-Cott2a	Diapaga	Cotton	Treated	16	2007	Q1 (100)
		BF-Cott3a	Lena	Cotton	Treated	18	2007	Q1 (100)
		BF-Cott4a	Diabo	Cotton	Treated	21	2007	Q1 (95), ASL (5)
		BF-Cott4b		Cotton	Treated	20	2009	Q1 (96), ASL (4)
		BF-Tom5a	Bobo ⁄ Kuinima	Tomato	Treated	33	2007	Q1 (42), ASL (58)
		BF-Mar5a		Marrow	Treated	31	2007	Q1 (65), ASL (35)
		BF-Okr5a		Okra	Treated	15	2007	Q1 (87), ASL (13)
		BF-Tob5a		Tobacco	Untreated	30	2007	Q3 (86), Q1 (7), ASL (7)
		BF-Cott6a	Bobo ⁄ Farako-BaˆCotton	Treated	15	2007	Q1 (93), ASL (7)
		BF-Cam7a	Ouagadougou	L.camara	Untreated	44	2007	Q3 (100)
		BF-Cam7b		L camara	Untreated	20	2009	Q3 (100)
		BF-Tom7a		Tomato	Treated	15	2007	Q1 (73), ASL (27)
	Benin	Be-Cott1a	Tanwe´Cotton	Treated	10	2007	ASL (100)
		Be-Cott2a	Koui	Cotton	Treated	18	2007	ASL (100)
		Be-Cott3a	Adjanoudoho	Cotton	Treated	9	2007	ASL (100)
		Be-Tom4a	IITA	Tomato	Treated	10	2007	ASL (100)
		Be-Cott5a	CRA-CF ⁄ Bohicon	Cotton	Treated	10	2007	ASL (100)
		Be-Cott5b		Cotton	Treated	11	2009	ASL (100)
		Be-Tom6a	Se´me´Tomato	Treated	16	2007	ASL (100)
		Be-Cas7a	Agonkanmey	Cassava	Untreated	20	2007	AnSL1 (4), AnSL2 (96)
		Be-Cott8b	Kandi Sina	Cotton	Untreated	21	2009	Q1 (4.8), ASL (95.2)
		Be-Cott9b	Kandi ⁄ Gamassosso	Cotton	Treated	18	2009	Q1 (5.5), ASL (94.5)
	Togo	To-Tom1a	Lome Ferme ⁄ UL	Tomato	Treated	13	2007	ASL (100)
		To-Cas2a	Lome Harbour	Cassava	Untreated	20	2007	AnSL1 (42), AnSL2 (58)
		To-Cott3b	Dapaong	Cotton	Treated	29	2009	Q1 (100)
		To-Cott4b	Tove´Cotton	Treated	8	2009	Q1 (8), ASL (92)

*The first two letters of the acronyms correspond to the country (BF, Burkina Faso; Be, Benin; To, Togo), the second part indicates the host plant and the number refers to a specific locality. The a and b samplings were carried out in 2007 and 2009 respectively. This column indicates whether pesticides are used or not. àNumber of individuals in the sample. §Percentage of each biotype found at the collecting site.

Table 2 .

 2 PCR primers and conditions used in the study.

					Annealing
		Targeted			temperature (°C) ⁄
	Symbiont	gene	Primers	Primer sequences	product size (bp)	References
	Portiera	ADNr16S 28F1098 R	5¢-AGAGTTTGATMTGGTCAG-3¢	58 ⁄ 1000	Zchori-Fein and
				5¢-AAAGTTCCCGCCTTATGCGT-3¢		Brown (2002)
	Rickettsia	ADNr16S Rb-FRb-R	5¢-GCTCAGAACGAACGCTATC-3¢	58 ⁄ 900	Gottlieb et al.
				5¢-GAAGGAAAGCATCTCTGC-3¢		(2006)
	Hamiltonella	ADNr16S Hb-FHb-R	5¢-TGAGTAAAGTCTGGAATCTGG-3¢	58 ⁄ 700	Zchori-Fein and
				5¢-AGTTCAAGACCGCAACCTC-3¢		Brown (2002)
	Cardinium	ADNr16S CFB-FCFB-R 5¢-GCGGTGTAAAATGAGCGTG-3¢	56 ⁄ 400	Weeks et al. (2003)
				5¢-ACCTMTTCTTAACTCAAGCCT-3¢
	Wolbachia	wsp	81F415R	5¢-TGGTCCAATAAGTGATGAAGAAAC-3¢	56 ⁄ 700	Braig et al. (1998),
				5¢-AAAAATTAAACGCTACTCCA-3¢		Vautrin (2008)
	Arsenophonus ADNr23S Ars-23S-1	5¢-CGTTTGATGAATTCATAGTCAAA-3¢	60 ⁄ 600	Thao and
			Ars-23S-2	5¢-GGTCCTCCAGTTAGTGTTACCCAAC-3¢		Baumann (2004)
	Fritschea	ADNr23S Frit-FFrit-R	5¢-TGGTCCAATAAGTGATGAAGAAAC-3¢	60 ⁄ 600	Thao et al. (2003)
	bemisiae			5¢-GCTCGCGTACCACTTTAAATGGCG-3¢

Table 3 .

 3 Names and accession numbers of samples used for the phylogenetic tree.

	Sample	Locality	Country	Host plant	Biotype	Accession number
	BF-Tom5a-1	Bobo ⁄ Kuinima	Burkina Faso	Tomato	ASL	HQ908636
	BF-Tom5a-9	Bobo ⁄ Kuinima	Burkina Faso	Tomato	ASL	HQ908637
	BF-Mar5a-1	Bobo ⁄ Kuinima	Burkina Faso	Marrow	ASL	HQ908638
	BF-Mar5a-6	Bobo ⁄ Kuinima	Burkina Faso	Marrow	ASL	HQ908639
	BF-Mar5a-8	Bobo ⁄ Kuinima	Burkina Faso	Marrow	ASL	HQ908640
	BF-Okr5a-3	Bobo ⁄ Kuinima	Burkina Faso	Okra	ASL	HQ908641
	BF-Cam7a-8	Ouagadougou	Burkina Faso	L. camara	Q3	HQ908642
	BF-Cam7a-9	Ouagadougou	Burkina Faso	L. camara	Q3	HQ908643
	BF-Cam7a-11	Ouagadougou	Burkina Faso	L. camara	Q3	HQ908644
		Ouagadougou	Burkina Faso	L. camara	Q3	HQ908645
	BF-Cam7a-13	Ouagadougou	Burkina Faso	L. camara	Q3	HQ908646
	Be-Tom6a-3	Se´me´Benin	Tomato	ASL	HQ908647
	Be-Cas7a-2	Agonkanmey	Benin	Cassava	AnSL1	HQ908648
	Be-Cas7a-7	Agonkanmey	Benin	Cassava	AnSL1	HQ908649
	To-Tom1a-2	Ferme ⁄ UL	Togo	Tomato	ASL	HQ908650
	To-Cas2a-1	Harbour zone	Togo	Cassava	AnSL2	HQ908651

AnSL, Sub-Saharan Africa non-Silver Leafing; ASL, Sub-Saharan Africa Silverleafing; Lantana camara, L. camara.

Ó 2012 The Authors Insect Conservation and Diversity Ó 2012 The Royal Entomological Society, InsectConservation and Diversity, 6,[411][412][413][414][415][416][417][418][419][420][421]

Acknowledgments

The authors thank Agence Universitaire de la Francophonie (AUF) for the Post-Doctoral fellowship of Olivier Gnankineá nd the collaboration established between Ouagadougou Uni-versity (Burkina Faso) and Lyon1 University (France). This research was supported by Agence Nationale de la Recherche (ANR).


Amanpreet Singh

Pushap Raj

Jan Dubowski

Narinder Singh

ATP Induced Modulation in π-π Stacking Interactions in Pyrene Based Zinc Complexes: Chemosensor Study and Quantitative Investigation of Apyrase Activity

come

Fluorescent zinc complexes of 1,2-disubstituted benzimidazole have been synthesized and characterized using single crystal XRD. The ligands were found to be less emissive due to photoinduced electron transfer (PET) mechanism originated from the electron pair of benzimidazole nitrogen. The complexation of ligands with Zn(II) not only enhances the fluorescent intensity, however also orient the ligands to an new packing. It was observed that the aromatic unit plays a decisive role in the packing of the molecules. The complex has extended the coordination through C-H interaction, whereas complex involved C-H interaction and C-H Br interaction for packing in supramolecular architecture. Among these complexes, showed most interesting noncovalent interaction pattern involving C-H interaction, C-H Br interaction and -stacking between pyrene rings. These noncovalent interactions govern photophysical properties that are sensitive toward microenvironment. Thus, by altering these interactions, the selective sensing for a particular analyte can be achieved. The complexes and have shown enhanced emission intensity upon interacting with ATP competitively in the presence of some other tested anions. A ratiometric change in emission spectra of the complex was observed upon binding with ATP in semi-aqueous medium offering the lowest detection limit of 15 nM. Upon interaction with the stacking between pyrene rings breaks and results in a decrease in excimer emission at 470 nm and increases in monomeric emission intensity at 410 nm. The AFM images of receptor show that upon addition of ATP to the solution, solvent mediated aggregation takes place, which results in the ratiometric detection. In the DMF solvent system aggregates were formed, whereas in Water:THF solvent system clear solution was converted to a highly viscous gel. To investigate the applications of the prepared sensor, fluorescence response of Hela cells enriched with ATP was observed using fluorescence microscopy. The fluorescence modulation of sensor in living cells makes the receptor practically applicable in a biological environment. Quantitative analysis of apyrase activity has shown that the presented sensor is capable to monitor the hydrolysis process in the biological system.

Fluorescent sensor, benzimidazole, pyrene, -stacking, ratiometric, adenosine triphosphate selective sensor can be developed. Recently Maji have developed a selective sensor for Al(III), that responds toward Al(III) by fluctuating emission behavior due to change induced by noncovalent interactions. 45 ATP has adenosine subunit that can interact through interaction with analyte. [46][47][START_REF] Kumar | Synthesis and Photophysical Properties of Fluorescence Molecular Probe for Turn-ON-Type Detection of Cytosine Bulge DNA[END_REF] Similarly three negatively charged phosphate units have a tendency to interact through ionic interaction.

In view of this, Yoon had constructed a dipodal receptor that contains pyrene excimer as signaling unit which upon stacking with adenine gave change in emission. [START_REF] Xu | Unique Sandwich Stacking of Pyrene-Adenine-Pyrene for Selective Ratiometric Fluorescent Sensing of ATP at Physiological pH[END_REF] The imidazolium unit conjugated to pyrene act as a phosphate ion receptor. The sensor showed ratiometric sensing of ATP, however, some analytes such as AMP and ADP also show some interference. Kataev have synthesized two copper complexes labeled with fluorescent coumarin and anthracene units for detection of ATP in an aqueous medium, which shows some interference due to other analytes. [START_REF] Kataev | Fluorescence Detection of Adenosine Triphosphate in an Aqueous Solution Using a Combination of Copper(II) Complexes[END_REF] Also, some binuclear complexes have been developed for sensing of ATP, AMP and pyrophosphates. The two phosphate units coordinate with two different zinc ions, resulting in a change in photophysical properties of the receptor.

In the present work, we have synthesized 1,2 disubstituted fluorescent labeled benzimidazole derivatives and their complexes with zinc ion. Particularly, zinc ion was chosen due to its configuration, which resists the loss of fluorescence upon complexation due to non-availability of open shell effect. The complexes and contain two halide ion and one solvent molecule which can be easily replaced by some strong field ligand. The most interesting features of these complexes are their differences in noncovalent interactions; resulting from a change in aromatic unit attached with benzimidazole moiety. These complexes have the ability to interact with analyte through ionic interaction as well as stacking. Due to the dual interaction of analyte, ratiometric sensing can be achieved. The ratiometric sensors are more reliable than simply "on-off" fluorescence sensors because the ratiometric behavior is independent from environmental factors, such as temperature, concentration, slit width and scan rate. 46 To the best of our knowledge, the prepared metal complexes of 1,2 disubstituted benzimidazole are the novel and there is no report on disubstituted benzimidazole based sensor for biomolecules. Thus, this system provides a simpler and more effective way to detect ATP than the methods reported in the existing literature. [47][START_REF] Kumar | Synthesis and Photophysical Properties of Fluorescence Molecular Probe for Turn-ON-Type Detection of Cytosine Bulge DNA[END_REF][START_REF] Xu | Unique Sandwich Stacking of Pyrene-Adenine-Pyrene for Selective Ratiometric Fluorescent Sensing of ATP at Physiological pH[END_REF] The interesting fact about this work is the formation of metallogel upon interaction of with ATP in THF: water (30:70).

The ligand and were prepared by a reported method from our research group. [START_REF] Sharma | Synergetic Catalytic Effect of Ionic Liquids and ZnO Nanoparticles on the Selective Synthesis of 1,2-Disubstituted Benzimidazoles Using a Ball-Milling Technique[END_REF] The ligand has been synthesized through a condensation reaction between one equivalent of o-phenylenediamine and two equivalent of the corresponding aldehyde in methanol. Characterization of the ligand was achieved using 1 H-NMR, [START_REF] Singh | Urea Based Organic Nanoparticles for Selective Determination of NADH[END_REF] C-NMR, and elemental analysis. 1 H-NMR signal at 5.8 ppm (singlet) confirmed the formation of the product, as this signal corresponds to aliphatic protons, showing intensity for two protons. Also, a number of protons in aromatic region of 1 H-NMR spectra and number of carbons in [START_REF] Singh | Urea Based Organic Nanoparticles for Selective Determination of NADH[END_REF] C-NMR spectra confirmed the structure of corresponding ligands. The complexes of ligand -with zinc ion were prepared in tetrahydrofuran and dimethylformamide (90:10). For complex , 2.0 equivalent of was dissolved in THF along with one equivalent of Zinc bromide. The dark powder was separated out that was dissolved by addition of 10% of dimethylformamide. The reaction mixture was heated for 3 hours and then cool down to room temperature. The dark brown colored crystals were separated out after 6 hours. The complex was prepared by reacting one equivalent of and one equivalent of Zinc chloride in THF: DMF (90:10). Similarly, complex was prepared using similar method, the one which was used for the synthesis of . All of the complexes were characterized using elemental analysis and infrared spectroscopy. For the final validation of structures, single crystal structure was obtained, showing 2:1, 1:1, and 1:1 composition of ligand: metal complexes, respectively for , and .

Crystal suitable for X-ray diffraction studies were isolated by slow evaporation technique in THF: DMF.

The crystals [{L1}ZnBr 2 ] ( ), [{L2(DMF)}ZnCl 2 ] ( ) and [{L3(DMF)}ZnBr 2 ] ( ) aree quite stable, therefore data of all crystals were collected at room temperature. Data collection and unit cell parameters are shown in table , whereas the list of bond length and bond angles is presented in table

to respectively in the supporting information. [START_REF] Bourhis | The Anatomy of a Comprehensive Constrained, Restrained Refinement Program for the Modern Computing Environment -Olex2 Dissected[END_REF] The structure was solved with the olex2.solve [START_REF] Bourhis | The Anatomy of a Comprehensive Constrained, Restrained Refinement Program for the Modern Computing Environment -Olex2 Dissected[END_REF] structure solution program using Charge Flipping and refined with the olex2.refine refinement package using Gauss-Newton minimization. Interestingly, upon changing the number of rings from benzene to naphthalene and pyrene, packing of the crystals changes dramatically. The angle of the plane between benzimidazole ring and another aromatic subunit greatly affect the absorbance and emission profile of complexes. Arguably, the most important feature of this paper concerns observation of the difference in non-covalent interaction resulting from a change in aromatic unit attached with benzimidazole moiety, which plays a decisive role in supramolecular packing as well as photophysical properties.

Crystal structure of complex (A) (B) and (C) with % probability thermal ellipsoids.

Crystal data and structure refinement hydrogens (Methylene and aromatic) to extend this 3-D supramolecular architecture. This kind of noncovalent interactions highly depends upon the aromatic surface, electron rich or electron deficient aromatic system. [START_REF] Morita | Synthetic Organic Spin Chemistry for Structurally Well-Defined Open-Shell Graphene Fragments[END_REF][START_REF] Chi | π-π Stacking and Ferromagnetic Coupling Mechanism on a Binuclear Cu(II) Complex[END_REF] The complex contained highly conjugated pyrene ring system that has completely different kind of noncovalent interactions involving -stacking between pyrene rings along with C-H and Br H interactions ( ). These weak noncovalent interactions are highly sensitive toward microenvironment and play a decisive role in photophysical properties; [START_REF] Mobin | Vapor-Diffusion-Mediated Single Crystal-to-Single Crystal Transformation of a Discrete Dimeric copper(II) Complex to a Discrete Tetrameric copper(II) Complex[END_REF][START_REF] Mobin | Single-Crystal to Single-Crystal Transformations in Discrete Hydrated Dimeric Copper Complexes[END_REF][START_REF] Vij | Covalent versus Charge Transfer Modification of Graphene/Carbon-Nanotubes with Vitamin B1: Co/N/S-C Catalyst toward Excellent Oxygen Reduction[END_REF][START_REF] Georgakilas | Noncovalent Functionalization of Graphene and Graphene Oxide for Energy Materials[END_REF][START_REF] Sivaraman | Development of a Pyrene Based "turn On" Fluorescent Chemosensor for Hg 2+[END_REF][START_REF] Vidya | Diverse Benzothiazole Based Chemodosimeters for the Detection of Cyanide in Aqueous Media and in HeLa Cells[END_REF][START_REF] Sivaraman | Pyrene Based Selective-Ratiometric Fluorescent Sensing of Zinc and Pyrophosphate Ions[END_REF] therefore there are reasonable possibilities to develop the sensors for biomolecules. Till date, many metal-organic complexes have been reported with -stacking interactions between pyrene ring or with another fluorophores. [START_REF] Mandal | Studies on Ruthenium Complexes of Pyrene-Appended Schiff Base Ligands[END_REF][START_REF] Gonell | Pyrene-Based Mono-and Di-N-Heterocyclic Carbene Ligand Complexes of Ruthenium for the Preparation of Mixed Arylated/alkylated Arylpyridines[END_REF][START_REF] Maity | Ru(II) and Os(II) Complexes Based on Terpyridyl-Imidazole Ligand Rigidly Linked to Pyrene: Synthesis[END_REF] However few reports are available where such kind of complexes are used for molecular recognition. [START_REF] Chong | Covalent Locking and Unlocking of an Atropisomeric Molecular Switch[END_REF][START_REF] Dial | Proton Grease: An Acid Accelerated Molecular Rotor[END_REF] Therefore to explore the possibility to interact these metal complexes with some particular analytes, molecular recognitions studies were performed with various anionic species using fluorescence spectroscopy. We expected that these specific orientations of metal complexes would introduce interesting photophysical properties that could be altered by the interaction with specific analytes.

Structural details of , showing various non covalent interactions (B) Structural details of

, showing various non covalent interactions (C) C-H•••Cl interaction leads to the formation of polymeric structure in the complex. (D) Fingerprint plots of the title complex: full and resolved into all, C H/H C, X H/H X and Cπ Cπ contacts showing the percentages of contacts contributed to the total Hirschfeld surface area of molecules.

To clarify the nature of intermolecular interactions, the Hirschfeld surface of the molecular moiety was analyzed which provides fingerprint of the interactions involved at intermolecular level. Hirshfeld surface mapped with norm takes various parameters into consideration to calculate normalized contact distance We have discovered that the major proportion of the Hirshfeld surface was compromised by Initially, spectroscopic properties of ligand (10 M) responding to Zn(II) were examined in HEPES buffer system (pH= 7.0, 1mM, DMF/Water 30:70). Absorbance spectra of have shown two absorbance band at 225 nm and 290 nm corresponding to to * and n to * transitions, respectively. As displayed in , absorbance intensity of ligand changes significantly upon addition of zinc ion. [START_REF] Liang | Zn(II) Complex of Terpyridine for the Highly Selective Fluorescent Recognition of Pyrophosphate[END_REF][START_REF] Isaia | Zinc(ii)-Methimazole Complexes: Synthesis and Reactivity[END_REF] To find out the molar excitation coefficient, absorbance was recorded by successive addition of Zn(II) to the solution of and . The molar absorbance coefficient was measured and summarized in Table 2. The modulation of molar absorbance coefficient can be seen upon substitution of a new aromatic ring to ligand . The emission spectra of the ligand show emission maxima at around 433 nm ( ex at 290 nm, scan rate 200 and slit widths = 10 nm). As the concentration of zinc ion in solution rises, the emission intensity at 430 nm increases which is related to the cancellation of the PET mechanism caused through the lone pair of the nitrogen donor ( ). The solid state structure clarify that ligand coordinates with zinc ion in 2:1 ( : Zn 2+ ) ratio. Further to find out the stoichiometry of complex formation in a solution state, measurement were performed through Job's plot method. A nonlinear fitting showed a maximum value at 0.66 which allows us to conclude that alike solid state structure, in solution state stoichiometry complex remain same 2:1 ( : Zn 2+ ). Further, binding constant of complexation was calculated using the Benesi-Hildebrand method. The inverse of the concentration of zinc ion was plotted against the inverse of change in emission intensity. The slope and intercept of the curve were determined using a linear regression method, which allowed determining the binding constant equal to 1.4 × 10 5 M -1 . To check the working pH for binding studies, emission profiles were recorded at different pH, suggesting that receptor stabilizes at pH 4 to 9 pH. At basic pH, zinc hydroxide was formed and thus causing decomplexation and loss of fluorescence due to restore of PET channel.

C•••H/H•••C interactions in each complex
Therefore all binding experiments were performed at pH 7.5.

The absorbance spectra of ligand has shown three absorbance maxima at 215nm, 245 nm and 295 nm ( ), that correspond to the presence of two different chromophores in the system. Upon addition of zinc chloride, the absorbance intensity of the receptor (~ 215 nm) changes significantly. The emission spectra of ligand were taken in DMF: water (30:70) ( ex at 290 nm, scan rate 200 and slit widths = 10 nm). The emission spectrum of the complex has shown two peaks at 425 nm and 550 nm ( ). As the concentration of zinc ion increases in a solution of complex , fluorescence intensity at 425 nm increases significantly whereas little enhancement was observed at 550 nm. The loss of emission due to photo induced electron transfer was inhibited through the coordination of lone pair of benzimidazole with Zn(II). For , stoichiometry and binding constant of complex in solution state were calculated using Job's plot and Benesi-Hildebrand curve. The stoichiometry of complex found to be 1:1 which perfectly matches that of the solid state structure and the binding constant was found to be 6.89 x 10 4 M -1 .

Due to the different electronic arrangement and orientation, three metal complexes offer anion binding pattern; which is unique to the structure of receptor with anionic species. The fluorescence intensity of each complex was recorded in presence of anions (F -, Cl -, Br ) and ATP, AMP, and ADP.

The complex showed enhancement in emission intensity upon interaction with ATP along with some interference with AMP and phosphate ( ). This enhancement in emission intensity attributes toward replacement of bromide with phosphate ion. Zinc ion has a tendency to bind strongly with a phosphate ion, [START_REF] Mesquita | Dinuclear Zinc(II) Macrocyclic Complex as Receptor for Selective Fluorescence Sensing of Pyrophosphate[END_REF][START_REF] Mikata | Fluorescent Detection of Phosphate Ion via a Tetranuclear Zinc Complex Supported by a Tetrakisquinoline Ligand and μ 4 -PO 4 Core[END_REF] while the orientation of a complex also provides the selectivity for a particular analyte. Binding studies of complex was performed using fluorescence spectroscopy with various anionic species ( ). Similarly for the complex , the studies also show fluorescence enhancement with different phosphate species. As shown in , ATP showed maximum fluorescence enhancement among investigated analystes. The stoichiometries of and complexes with ATP were evaluated using Job's Plot, which indicates the formation of a 1:1 complex between and ATP. The major drawback of the sensor was its non-specificity because it also shows emission enhancement with other phosphate ions ( ). Therefore it is not suitable for real time determination of ATP.

Photophysical properties of ligands and metal complexes abs (nm) ε 0 (L mol with an R 2 value of 0.9993 ( ) and detection limit of 15 nM. To analyze the effect of another ion on detection of ATP, the competitive binding experiments were performed, which showed that none of the interfering ions obstruct the detection of ATP ion. Also, the UV-visible absorbance spectra of the complex were recorded with the addition of ATP in the THF:Water (30:70) solvent system ( ).

Change in emission profile of ligand receptor and penetrate between two pyrene rings. [START_REF] Xu | Unique Sandwich Stacking of Pyrene-Adenine-Pyrene for Selective Ratiometric Fluorescent Sensing of ATP at Physiological pH[END_REF] Similar results were obtained from fluorescence spectroscopy. Upon addition of ATP, two types of interactions were involved between receptor and ATP. One is ionic interaction and another is stacking between Pyrene-Adenine-Pyrene sandwiches.

In this way, polymeric structure extended through hydrophobic stacking interaction was formed causing turbidity in the solution. The ratiometric change in emission ( ) and increase in turbidity indicate that the clear solution changes into the aggregates. To find out the morphology of aggregates formed upon addition of ATP, atomic force microscopic images (AFM) of receptor were taken in the presence of ATP. AFM imaging confirmed the formation of aggregates ( ).

Initially, the receptor was purely crystalline in nature. On the addition of 1.5 equivalent of ATP, aggregates were formed as also confirmed using DLS (Dynamic Light Scattering) experiments. The size of aggregates formed was determined using DLS which showed the particles size distribution in the range of 35 nm to 40 m ( ). To determine the stoichiometry of complex formation between ATP and complex , fluorescence data were utilized to construct Job's plots. The -axis of the Job's plot is labeled as the mole fraction of ATP ( ). The intersection of the two linear portions of the Job's plot gives the mole ratio corresponding to the binding stoichiometry between ATP and , which confirmed formation of the 1:1 complex between ATP and . The binding constant ( a ) of for ATP was also determined by constructing the Benesi Hildebrand plot; a a value of 2.57 (± 0.04) × 10 4 M -1 was obtained from the slope and intercept of the line ( ).

All the binding studies were performed in Water: DMF (70:30), while the solution of receptor turned turbid upon interaction with ATP. Further, we have examined the behavior of receptor with ATP in another solvent system. It was observed that in tetrahydrofuran:water (30:70), the more viscous liquid was formed which turned to metallogel upon cooling for 6 hours. To investigate the morphology of gel, scanning tunneling microscopy images were recorded and it was observed that a polymeric network was formed which quench the excimer fluorescence. As shown below in cartoon diagram, ATP interacts with probe in such a way that adenosine unit penetrate between two pyrene rings which abolish the -interaction between two pyrene rings and induced new sandwich complex having -stacking between pyrene-adenosinepyrene. Such kind of planar conjugated systems are well known for formation of -stacking induced gel. [START_REF] Lovitt | Coordination Chemistry of N-Picolyl-1,8-Naphthalimides: Colourful Low Molecular Weight Metallo-Gelators and Unique Chelation Behaviours[END_REF][START_REF] Gunnlaugsson | Supramolecular Pathways: Accessible Self-Assembly[END_REF][START_REF] Jones | Gels with Sense: Supramolecular Materials That Respond to Heat{,} Light and Sound[END_REF] This supramolecular assembly highly depends upon anionic species, nature of interaction and solvent system. [START_REF] Fang | Macroscopic and Fluorescent Discrimination of Adenosine Triphosphate via Selective Metallo-Hydrogel Formation: A Visual, Practical, and Reliable Rehearsal toward Cellular Imaging[END_REF][START_REF] Jung | Self-Assembled Tb 3+ Complex Probe for Quantitative Analysis of ATP during Its Enzymatic Hydrolysis via Time-Resolved Luminescence in Vitro and in Vivo[END_REF][START_REF] Bhattacharya | Pyrazole-Based Metallogels Showing an Unprecedented Colorimetric Ammonia Gas Sensing through Gel-to-Gel Transformation with a Rare Event of Time-Dependent Morphology Transformation[END_REF][START_REF] Ai | Cyclometalated Platinum(II) Complexes of 1,3-Bis(1--Butylpyrazol-3-Yl)benzenes: Synthesis, Characterization, Electrochemical, Photophysical, and Gelation Behavior Studies[END_REF] It is interesting that metallogel is formed in THF:Water (30:70), whereas aggregates are formed in DMF:Water. Therefore the gel formation is not only selective for anionic species (ATP) but also solvent decide the morphology formed in the solution.

To investigate the interference caused by competing analytes, such as ADP, AMP, PPi, NADH and other anions, we recorded the corresponding fluorescence spectra.

It was find out that ADP and AMP show negligible change in monomeric and excimer emission peaks.

Similarly, other analytes did not show any significant change in emission profile. As shown in , the ratio (I monomer /I excimer ) for ATP is much larger as compared to other analytes. The above selectivity for ATP can be explained on the basis of the binding pattern. ATP bind with zinc ions through ionic bond by replacing two bromide ions and, similarly, AMP and ADP bind with receptor through ionic bond.

However hydrophobic interaction ( stacking) plays a crucial role in emission change. Therefore due to the difference in chain length in ATP, AMP and ADP, these analytes behave differently while interacting with receptor . For perfect binding or ratiometric sensing, it is necessary that both interactions (ionic and hydrophobic) occur simultaneously, which is possible only in case of ATP. Thus, neither AMP nor ADP shows response to receptor . Further to validate the observation, the powder XRD spectra of complex has recorded and compared with the simulated XRD pattern, which was perfectly superimposed ( ). Then the complex was reacted with ATP and ADP in THF: water, after removing the solvent with the vacuum, the prepared compound has washed with water, to remove the free phosphate species. The broad XRD pattern of R3-ATP revealed the formation of polymeric aggregates, 80 whereas solid state XRD spectra of R3-ADP was showing sharp peaks, which also have similarity with XRD spectra of complex. To confirm the formation of R3-ATP complex, Raman spectra has recorded. To record the Raman spectra, the crude obtained from the reaction of Zinc complex R3 with ATP, has washed with distilled water to remove the unreacted Adenosine triphosphate ions. The Raman spectra of this crude expressively different pure complex and showing peaks corresponds to Zinc-phosphate bond and Phosphorous-oxygen bonds (725 cm -1 , 1005 cm -1 , 1122 cm -1 ) which confirm the formation of R3-ATP complex ( ). However, ADP ion was showing similar spectra as the complex , reveals that complex R3 is not reactive toward ADP. To confirm the complexation of ATP with complex R3, the chemical characterization of complex and R3-ATP has performed using energy-dispersive X-ray spectroscopy ( ). It revealed that the complex has Zn, Br, C, N and O; however, the bromide ion disappears upon complexation with ATP. The presence of peak at 2.1 corresponds to phosphorous, which confirmed the replacement of Br -ion with phosphate unit of ATP. Whereas, in crude of R3-ADP, the peak corresponds to Br still appear and negligible presence of phosphorous has seen. : To analyze the response of the receptor to intercellular investigation of ATP, HeLa cells were investigated under a fluorescence microscope for an excitation wavelength of 340 nm -380 nm and an emission wavelength of 435nm -485nm for blue fluorescence.

Cartoon

For the fluorescence microscopic experiments, HeLa cells were grown in Dulbecco's Modified Eagle's medium (DMEM) using reported protocol. [START_REF] Samanta | A Solo Fluorogenic Probe for the Real-Time Sensing of SO 3 2and SO 4 2-/HSO 4 in Aqueous Medium and Live Cells by Distinct Turn-on Emission Signals[END_REF] The cells were divided into two parts. Among these, one part was incubated with 10 M of oligomycin that is well known for its ATP syntheses inhibition activity. Then both parts were exposed to receptor (20 M) for 3 hours. Before the fluorescence measurement, the cells were washed with phosphate buffer solution (PBS). The receptor was causing a loss in fluorescence in oligomycin part, whereas HeLa cell incubated with only probe show emission in HeLa cells ( ). These experiments showed that receptor is capable to determine ATP in presence of another inorganic phosphate such as AMP and ADP in the intercellular system. Further, the ratiometric emission conversion with ATP was used to explore the probe to study the activity of apyrase enzyme that is well known for hydrolysis of nucleotide triphosphates. Our probe show ratiometric response only with ATP, therefore upon hydrolysis signature emission profile of -ATP complex should disappear. With this in mind, a solution of (20 M), ATP (20 M) and apyrase was prepared at pH 6 and emission profile was recorded after a short interval of time.

The curve ( ) shows that in the absence of apyrase, fluorescence intensity (F 410nm /F 470nm ) remains almost unchanged, however in the presence of two different concentrations of apyrase it decreases dramatically, which validate the conversion of ATP into AMP and ADP and finally into phosphates. To explore the mechanism more deeply, the turbidity of the solution was measured with time, which also shows a continuous decreas with time (up to 60 minutes) ( ). Thus, the prepared probe can be used for monitoring the activity of apyrase enzyme.

(A) Competiting binding of complex with various anionic species. (B) Change in fluorescence life time of upon interaction with ATP.

Fluorescence is perfect nano scale probe as its decay could take place on the nanosecond level.

Therefore it is a promising technique to evaluate molecular interactions and changes in a nanoenvironment. To analyze the effect of aggregation on photophysical properties and decay time, time resolved fluorescence spectra were recorded at the wavelength of 472 nm, which enlightens the mechanism of sensing of ATP. Fluorescence quantum yield and life time were determined using time resolved fluorescence and radiative decay constant using the following formulas: and where ᶲ f is fluorescence quantum yield, τ is a life time, is radiative decay constant and is nonradiative decay constant.

The rate of radiative decay of the excited state decreases as the concentration of ATP increases in solution, whereas life time of excited state increases. This transformation is due to the formation of aggregates where properties of the excited state are different and lead to an increased life time. [START_REF] Kumar | Water Dispersed Fluorescent Organic Aggregates for the Picomolar Detection of ClO 4 in Water, Soil and Blood Serum and the Attogram Detection of ClO 4 in the Solid State by a Contact Mode Method[END_REF] Whereas a decreasing radiative decay constant is attributed to quenching of the excimer emission. We observed a minor change in the nonradiative decay constant upon addition of ATP to the solution.

In summary, we have synthesized three zinc complexes tagged with fluorescence moieties that can form a tertiary complex with biologically important phosphates. Upon addition of ATP, the complex showed distinct spectroscopic behavior due to the replacement of bromide ion. The and interact only through ionic interaction with phosphates. Therefore, these complexes show emission enhancements with ATP along with some interference of AMP and phosphate. The complex has a tendency to interact through by both stacking and ionic interaction. It selectively binds with ATP even in the presence of other phosphates. Two types of interactions act differently on emission properties of the complex. Ionic interactions cause enhancement in fluorescence intensity at 410 nm, whereas the stacking interaction between pyrene and adenosine ring quenches the emission at 470 nm. Thus, the pyrene conjugated complex of Zinc ( is capable of detecting ATP ratiometrically with a low detection limit. Furthermore, the complex was explored for monitoring the activity of Apyrase enzyme during (?) hydrolysis of ATP. The outcome of this work is important for the development of a highly selective ATP sensor using ionic and other non-covalent interactions.

Ortho-phenylenediamine, aldehydes, and biomolecules (Analytical grade) were procured from Sigma Aldrich and used without further purification. Oligomycin (from Streptomyces diastatochromogenes) and Apyrase enzyme (From Potato) was purchased from Sigma Aldrich. and ADP (Adenosine diphosphate) (1mM) were prepared in distilled water. The pH of the solution was maintained at 7.0 using 1mM HEPES buffer solution. [START_REF] Sharma | Synergetic Catalytic Effect of Ionic Liquids and ZnO Nanoparticles on the Selective Synthesis of 1,2-Disubstituted Benzimidazoles Using a Ball-Milling Technique[END_REF] In a round bottom flask, the benzaldehyde (2.44 gm, 10 mmol) and o-phenylenediamine (1.08 gm, 10 mmol) was dissolved in 50 mL of methanol along with (0.185 gm, 0.5 mmol) of zinc perchlorate. The reaction mixture was refluxed for 8 hours; and the progress of the reaction was monitor using TLC. After completion of the reaction, the mixture was allowed to cool to room temperature.

  that were the highest in case of the complex. The portion of the Hirshfeld surface corresponding to Cπ••• Cπ increases as the number of aromatic rings increased from benzene to naphthalene and further to pyrene. These results validate the ability of pyrene ring for the formation of supramolecular self-assembly through π•• π stacking.

  Photoinduced Electron Transfer mechanism. On complexation, the lone pair of benzimidazole ring causes the fluorescence quenching due to the PET phenomenon transferred to the zinc ion. However, the emission intensity at 470 nm enhanced due to the formation of excimers. To confirm the binding of Zn(II) with ligand , the titration was performed by successive addition of zinc ion into 10 M solution of complex . As shown in figure , the orientation of ligand changes on complexation with zinc ion in such way that all the pyrene rings got stacked and gave strong excimer emission band at 470 nm. The binding constant for complex calculated using Benesi-Hildebrand method. A graph has plotted between inverse of concentration of analyte and inverse of change in fluorescence ( ). The binding constant has calculated from intercept and slope of the curve, which has found to be 7.34 x 10 4 .

(

  10 M) upon addition of 15 M of Zinc bromide in DMF: water (30:70) HEPES buffer (B) Packing of complex , showing stacking interaction between pyrene rings. (C) Change in emission profile of complex (10 M) upon addition of 16 M of various anions in HEPES buffer (DMF: water) (d) Change in emission profile of complex with successive addition of ATP (0-16 M). To find out the stoichiometry of complexation, the Job's plot has constructed. The plot between mole fraction of ATP and [HG], shows maximum value for [HG] at a mole fraction of 0.5 which indicates the 1:1 complexation between and ATP. The changes induced in excimer and the monomeric emission peak of the receptor indicate the breakdown of stacking ( ); also the clear solution converts into a highly viscous liquid which formed a luminescent metallogel upon cooling. Earlier Yoon, have studied the Sandwich Stacking of Pyrene-Adenine-Pyrene for determination of ATP. Here adenine ring breaks the stacking in

  diagram of ATP induced gel formation. (B) AFM images of upon interaction with ATP in THF: water (C) AFM images of upon interaction with ATP in DMF: water (D) The reaction showing the hydrolysis of ATP in presence of apyrase enzyme. (E) Change in turbidity of + ATP solution in DMF: water in presence of apyrase with time. (F) Change in turbidity of + ATP solution in DMF: water in presence of apyrase (0 to 250 mU) with time.

  Methanol and tetrahydrofuran were purchased from Merck. The photoluminescence experiments were performed on Perkin Elmer LS-55 instrument at a constant scan speed of 200. For all experiments, excitation and emission slit width was kept 10 nm. 1 H-NMR and the[START_REF] Singh | Urea Based Organic Nanoparticles for Selective Determination of NADH[END_REF] C-NMR spectrum were recorded on Jeol instrument which was operated at 400 MHz for 1 H-NMR and 100 MHz for[START_REF] Singh | Urea Based Organic Nanoparticles for Selective Determination of NADH[END_REF] C-NMR. The Single crystal data for three complexes were measured on Bruker X 8 A P E X I I I K A P P A C C D diffractometer at room temperature using graphite monochromatized Mo-K radiation (λ = 0.71073 Å). Elemental analyses were carried out using Fisons CHNS analyzers. The morphology of gel was found out on a scanning electron microscope (SEM JEOL JSM-6610LV) using a voltage of 15 KV and Atomic force microscopy (AFM). The particle size of nanoparticles was determined by dynamic light scattering (DLS) using external probe feature of Metrohm Microtrac Ultra Nanotrac particle size analyzers. Fluorescence microscopy of HeLa cells was recorded on Leica fluorescence microscope.Fluorescence life time was calculated from time resolved fluorescence spectroscopy using PicoQuant fluorescence spectrophotometer.The stock solution of each ligand (10 M) was prepared in DMF: water(30:70). Similarly, a stock solution of zinc bromide and zinc chloride (each 1mM) was prepared in hot distilled water and diluted further as a requirement of the experiment. The solution of various anions as tetra butyl ammonium salt (F -, Cl -, Br -, I -, CH 3 COO -) and ATP (Adenosine triphosphate), AMP (Adenosine monophosphate),

(

  20 mL) along with ligand L1 (200 mmol). The reaction mixture was heated and stirred for 20 minutes.The color of the solution becomes more intense. The solution was kept undisturbed for 24 hours for slow evaporation. Dark brown needle shaped crystals were formed that were suitable for single crystal data collection. Elemental analysis: Calculated: C, 62.01; H, 3.99; N, 5.04; Found: C, 61.95; H, 3.95; N, 5.00. The Supporting Information is available free of charge on the ACS Publications website at DOI. Absorption and emission properties, 1 H-NMR,[START_REF] Singh | Urea Based Organic Nanoparticles for Selective Determination of NADH[END_REF] C-NMR, Bond angles and bond length of complex, DLS, Fluorescence microscopy images. Dr. Narinder Singh, a Department of Chemistry, Indian Institute Technology Ropar, Punjab, 140001, India This work was supported by research grant from Indo-Canada project sponsored by DBT-New Delhi and IC-IMPACTS Canada. A. S. and P. R. are thankful to CSIR-New Delhi (9/1005(0010)/2014-EMR-1) and UGC-New Delhi respectively for their fellowship. (1) Gale, P. A.; Caltagirone, C. Anion Sensing by Small Molecules and Molecular Ensembles. , (13), 4212-4227 DOI: 10.1039/C4CS00179F. (2) Gong, Z.-L.; Zhong, Y.-W. H 2 PO 4 --and Solvent-Induced Polymorphism of an Amide-Functionalized [Pt(N^C^N)Cl] Complex. , (20), 10143-10151 DOI: 10.1021/acs.inorgchem.6b01059. (3) Patra, C.; Bhanja, A. K.; Mahapatra, A.; Mishra, S.; Saha, K. Das; Sinha, C. Coumarinyl Thioether Schiff Base as a Turn-on Fluorescent Zn(II) Sensor and the Complex as Chemosensor for the Selective Recognition of ATP, along with Its Application in Whole Cell Imaging. , (80), 76505-76513 DOI: 10.1039/C6RA12369D.

(

  The orange crystalline solid was separated out which

	properties. Yield 91%, Elemental analysis: Calculated: C, 87.47; H, 5.24; N, 7.29; Found: C, 87.41; H,
	5.17; N, 9.27.
							The
	compound	was synthesized using same method as , 1-Pyrenecarboxaldehyde (4.96 gm, 10 mmole)
	was used instead of benzaldehyde and reaction mixture was refluxed for 10 hours. The orange solid was
	separated out after 10 hours of refluxing which constituted pure compound	w i t h f o l l o w i n g
	properties. Yield 91%, Elemental analysis: Calculated: C, 89.86; H, 4.90; N, 5.24; Found: C, 89.82; H,
	4.85; N, 5.18. 1 H NMR (400 MHz, DMSO-D6) δ 8.01 -7.94 (t, 4H,	), 7.86 -7.79 (t, 4H,
	), 7.69 (t, 1H,	), 7.61 (t, 2H,	), 7.57 (t, 2H,	), 7.51 (t, 2H,	), 7.45 (t,
	3H,	), 7.38 (t, 2H,	), 7.28 (t, 1H,	), 7.22 (t, 3H,	), 6.60 (d, 1H,	),
	5.83 (s, 2H). 13 C NMR (400 MHz, DMSO-D 6 ) δ 175.70, 152.80, 151.86, 144.40, 143.35, 135.87, 134.94,
	134.13, 133.71, 133.59, 132.46, 132.04, 131.00, 130.69, 130.29, 129.11, 128.92, 128.85, 128.73, 128.39,
	128.27, 127.84, 127.61, 126.99, 126.94, 126.87, 126.59, 125.91, 125.83, 125.53, 123.63, 123.38, 123.19,
	122.79, 122.13, 120.03, 119.60, 111.88, 111.79, 46.12.
			Zinc(II)bromide (100 mmol) was dissolved in methanol -tetrahydrofuran
	(20 mL) along with ligand L1 (200 mmol). The reaction mixture was heated and stirred for 20 minutes.
	The color of the solution becomes more intense. The solution was kept undisturbed for 24 hours for slow
	evaporation. Dark brown needle shaped crystals were formed that were suitable for single crystal data
	collection. Elemental analysis: Calculated: C, 60.52; H, 4.06; N, 7.06; Found: C, 60.47; H, 4.00; N, 7.01.
			Zinc(II)chloride (100 mmol) was dissolved in methanol -tetrahydrofuran
	(20 mL) along with ligand L1 (200 mmol) The reaction mixture was heated and stirred for 20 minutes.
	constituted pure compound The color of the solution becomes more intense. The solution was kept undisturbed for 24 hours for slow with following properties. Yield 94%, Elemental analysis: Calculated: C, evaporation. Dark brown needle shaped crystals were formed that were suitable for single crystal data 84.48; H, 5.67; N, 9.85; Found: C, 84.42; H, 5.61; N, 9.78. collection. Elemental analysis: Calculated: C, 62.70; H, 4.58; N, 7.08; Found: C, 62.67; H, 4.55; N, 7.02.
	compound	T h e , naphthaldehyde (3.44 gm, 10 mmoles) Zinc(II)bromide (100 mmol) was dissolved in methanol -tetrahydrofuran was synthesized using the same method as
	was used instead of benzaldehyde and reaction mixture was refluxed for 10 hours. The orange solid was
	separated out after 10 hours of refluxing which constituted pure compound	w i t h f o l l o w i n g

  4) Raj, P.; Singh, A.; Kaur, K.; Aree, T.; Singh, A.; Singh, N. Fluorescent Chemosensors for Selective and Sensitive Detection of Phosmet/Chlorpyrifos with Octahedral Ni 2+ Complexes. , , 4874-4883 DOI: 10.1021/acs.inorgchem.6b00332. Dong, L.; Zhang, J.; Li, J.; Jiang, L.; Huang, Y.; Qin, S.; Hu, C.-W.; Yu, X.-Q. Imidazolium-Functionalized BINOL as a Multifunctional Receptor for Chromogenic and Chiral Mu, L.; Zeng, X.; Zhao, J.-L.; Redshaw, C.; Ni, X.-L.; Yamato, T. An NBD-Armed thiacalix[4]arene-Derived Colorimetric and Fluorometric Chemosensor for Ag + : A Metal-Ligand Yang, Y.; Chen, S.; Ni, X. Fluorescent Chemosensor for Nitrate in Acidic Aqueous Solution and. , (14), 7461-7466 DOI: 10.1021/acs.analchem.5b01774. (11) Zhao, J.; Yang, D.; Zhao, Y.; Cao, L.; Zhang, Z.; Yang, X.-J.; Wu, B. Phosphate-Induced

	(5)	Singh, A.; Singh, A.; Singh, N.; Jang, D. O. A 2-Mercaptobenzimidazole-Based Emissive Cu(I)
		Complex for Selective Determination of Iodide with Large Stokes Shift.
		,	, 372-379 DOI: 10.1016/j.snb.2016.11.157.
	(6)	Lu, Q.-S.; Anion Recognition.	,	(3), 669-672 DOI: 10.1021/ol8027303.
	(7)	Fu, Y.; Receptor of Anions.		,	(10), 3552-3560 DOI: 10.1039/c2dt32115g.
		Experimental and Theoretical Investigations.	,	(15), 4484 DOI:
		10.1039/c2dt12126c.
	(9)	Singh, A.; Raj, P.; Singh, N. Benzimidazolium-Based Self-Assembled Fluorescent Aggregates for
		Sensing and Catalytic Degradation of Diethylchlorophosphate.	,
		, 28641-28651 DOI: 10.1021/acsami.6b09983.
	(10) Fluorescence of a Tetraphenylethene-Substituted Tripodal Tris(urea) Receptor.	,
		(17), 7360-7365 DOI: 10.1039/C6DT00672H.
	(12) Jia, C.; Zuo, W.; Zhang, D.; Yang, X.; Wu, B. Anion Recognition by Oligo-(Thio)urea-Based
		Receptors.

(8) Kundu, T.; Chowdhury, A. D.; De, D.; Mobin, S. M.; Puranik, V. G.; Datta, A.; Lahiri, G. K. Selective Recognition of Fluoride and Acetate by a Newly Designed Ruthenium Framework: , , 9614-9627 DOI: 10.1039/C6CC03761E.