COCCINELLID PREDATORS OF APHID AND THEIR PHYLOGENETIC ANALYSIS USING COI GENE SEQUENCES

An extensive survey of aphid predatory Coccinellid beetles (Coleoptera: Coccinellidae) was conducted in Jahangirnagar University campus, Savar during October 2014 to March 2016. Total nine ladybird beetles viz, Adalia bipunctata, Coccinella septempunctata, Coccinella transversalis, Cycloneda munda, Harmonia axyridis, Hippodamia convergens, Micraspis discolor, Scymnus nebulosus, Sticholotis sp. were identified as predators of aphid. Among them, Micraspis discolor was most abundant predator. Biology and bio-control potential of M. discolor was studied in laboratory condition on bean infesting aphid (Aphis fabae). The maximum predation was 102±1.83 observed in 4 instar larvae of M. discolor. Along with biological study, phylogenetic relationship of ladybird beetles was constructed based on mitochondrial COI gene. Out of nine identified coccinellids, five species showed more conserve region than the rest in alignment. The interspecific genetic distance ranges for nine beetles were very low (0.15-0.24). Higher interspecific genetic diversity recorded for H. axyridis. The phylogenetic tree was created and analyzed using both Neighbour Joining and Maximum likelihood methods to define the origin and evolutionary relationships of the species. Molecular analysis demonstrated that evolution of these species has been occurred from a common ancestor. Such study of ladybird beetles would be helpful in biological control program of aphid pest.


Introduction
Aphids are broadly recognized as some of the most destructive pests and its distribution is worldwide. Approximately 4000 species of aphids have been described suckling over 250 agricultural and horticultural crops throughout the world (Ali and Rizvi, 2007). Black bean aphid (Aphis fabae, Homoptera: Aphididae) is the most domineering pest of beans and some other crops. Bean aphid invades a large number of fruit, vegetable, agronomic, and ornamental plants. This pest accounts for yield losses ranging from 37 to 90% (Abate et al., 2000;Ampofo and Massomo,2009). They cause impairment directly by sucking cell sap, secrete honeydew resulting in development of sooty mold on leaves and shoots and secondarily as vectors of certain plant viruses (Difonzo et al., 1997;Kennedy et al., 1962;Raboudi et al., 2002).
Till now, using insecticide is the only option for the farmers or gardeners to control this pest. But due to ban on harmful insecticides by WHO and FAO, carcinogenic effect, environmental pollution, resistance of pest to insecticide lead researchers to search for alternative safe control measures.
Predation is assumed to be one of the major biotic mortality factors dropping insect pest populations, and using them in insect pest management programs has been receiving augmented attention because of the current requirement to reduce the exclusive usage of insecticides for pest control (Luck, 1984;Riudavents and Castane, 1998;Sarmento et al., 2007). Ladybirds are universal predator that feed on a diverse range of foods. Many injurious insects have been successfully controlled by the Coccinellid beetles in agroecosystem (Agarwala et al., 1988). Therefore, an attempt was taken to study the predators of aphid and efficacy of the most effective one.
Mitochondrial genomes are gradually recognized as influential phylogenetic indicators for determining relationships at various hierarchical levels (Gillet et al., 2014;Simon and Hadrys, 2013).Phylogenetic analysis has great impact on classification, forensics, identifying the origin of pathogen and help in conserving nature.
Before deciding to take any pest control action, proper attempt to identification of insect using molecular identification technique would be very effective. This will ultimately help to reduce the risk of damage caused by Research Article F.F. Rain et al. (2016) Int J Appl Sci Biotechnol, Vol 4(3): 408-416 This paper can be downloaded online at http://ijasbt.org & http://nepjol.info/index.php/IJASBT insects. The purpose of this study was to construct the phylogenetic relationship within the ladybird beetles Coccinellidae using COI gene and estimate the divergence from the common ancestor.

Study area and preparation of experimental plots
Experiments were performed in the Insect Rearing and Experimental Stations (IRES), Savar having geographical location 23°52'32.28"N, 90° 16'0.06"E from October , 2014to March, 2016. This area has a good composition of vegetables, fruits, and ornamental trees. The ecological condition supports the prey-predator relationship because this area is free of chemical insecticide usage.
For conducting this research, few small plots of bean (Lablab purpureus) were prepared. In these small plots, chemical fertilizer or insecticides were not used to keep the insects undisturbed. In addition to this other bean gardens of this campus and surroundings were also observed to find any coccinellid beetles.

Coccinellid Predators of Bean Aphid and Their Identification
Aphids and its natural enemies were keenly observed in the field to make a list of natural enemies of aphid. Predators that were found to predate aphid in the field were collected and taken to the Laboratory of Molecular Entomology, Wazed Miah Science Research Center to study predatory efficacy in laboratory condition. Immature stages that were found to predate aphid were also reared in the laboratory condition to check the life cycle and get the adult stage, which is needed for proper identification. Identification was performed using proper keys (Blackburn,1892;Borror et al., 1981;Omkar and Bind,1993;Omkar and Bind, 1995;Omkar and Bind,1996;Omkar and Pervez,1999;Omkar and Pervez, 2000;Poorani, 2002).All natural enemies were not possible to identify up to species level due to lack of adequate and appropriate keys. In those cases samples were identified up to family or generic level.

Predatory Efficacy of Micraspis discolor in the Laboratory Condition
M. discolor were observed in the bean crop to feed aphid voraciously. Egg lying of this beetle was followed to collect eggs. Collected eggs were placed on a blotting paper in the petri plate (60 mm15 mm). Petri plates were then transferred to Laboratory. To study its life cycle and predatory efficiency of different life stages, eggs were placed in an incubator with prefixed temperature 28±2 0 C and light hour of 16 hours. After hatching, the 1st instar larvae were separated individually in different petri plates using soft camel hair brush. Larvae were offered a predetermined number of same sized bean aphids in every 24 hours as their food and their predatory efficacy were recorded. Thus, larval metamorphosis up to adult was observed. Predatory efficacies of the adults were also recorded in the same manner.

Data Analysis
Development and predation at different life stages of M. discolor was recorded with respect to aphid species. SPSS 16.0 software was used for data analysis. COI gene sequence of nine coccinellid beetle species was collected from National Center for Biotechnology Information (NCBI) for their molecular and phylogenetic analysis (http://ncbi.nlm.nih.gov) ( Table 1).

Sequence Alignment and Phylogenetic Analysis
COI sequences were aligned using ClustalW algorithm with the help of MEGA tools (version 6) with gap opening penalty 15, gap extensions penalty 6.66, transition weight 0.5 and delay divergent cutoff 30% (Kobayashi et al., 1998;Simon and Hadrys, 2013). Multiple sequence alignment images were prepared using jalview, version 2.9 (Von der Schulenburg et al., 2011). For calculation of genetic distances among sequences, we used Kimura's two parameter method (K2P) of base substitution in MEGA 6 (Tamura et al., 2013).
Preliminary phylogenetic analyses were performed with MEGA 6 using both neighbor joining (NJ) and maximum likelihood (ML) tree reconstruction methods (Tamura et al., 2013). The evolutionary history was inferred using the Neighbor-Joining (NJ) method (Saitou and Nei, 1987). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was shown next to the branches (Felsenstein, 1985). The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.
The evolutionary history was inferred by using the Maximum Likelihood (ML) method based on the Kimura 2-parameter model (Kimura, 1980).The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and

Coccinellid Predators of Aphid in the Field
Natural enemies are very important in the control of bean aphids that kill pests. Usually natural enemy populations did not appear in significant numbers until aphids begin to be numerous. In the present experiment, predators of the bean aphid were taken into consideration as natural enemies. Among the most important natural enemies were various predators that were found to feed on aphids. Coccinellid beetle's adults and larvae were most abundant. During the survey period, Coccilenellid ladybird beetles were found to predate aphids and nine (9) predatory beetles were identified (Table 2). Length (L)= 3.64 mm to 4.08 mm……………………………Micraspis discolor 8. Pubescent light reddish-brown species with irregular dark markings on the elytra; a dark median area of the pronotum; Length (L) = 2 to 2.2 mm……………………..…. Scymnus nebulosus 9. Pronotum with anterolateral angle delimited from disc by an oblique line; prosternum anteromedially with a reflexed lobe; maxillary palpus with apical palpomere conical or elongate-oval and pointed apically; postcoxal line short, divided and not forming an arc; Minute, Length (L) = 1.0 to 1.5 mm………………………….…… Sticholotis sp.

Density of Black Bean Aphid and Micraspis discolor
As Coccinellids are proven biological control agents, an attempt to count these predators in relation to aphid was taken. Infestation of Aphis fabae in bean plant was very low in early part of August but it increased gradually up to mid-September. The highest Aphid population was 108.67 per leaf in 9 September. In the meantime predators were also raised. Micraspis discolor were observed in the bean crop to feed aphid voraciously. The maximum predation 102±1.83 was observed by 4 th instar larvae of M. discolor followed by the 1 st instar, 2 nd instar and 3 rd instar during the year of study. The first instar larvae of M. discolor could consume 20 ± 0.41 aphids/day during investigation. The 2nd instar larvae could devour 39 ± 0.75. Similarly the corresponding values for the 3rd instar and 4 th instar larvae were observed as 60.5 ± 2.57, 102±1.83 respectively. The total and per day consumption of the aphids by the different instars of this grub was found significant (Table 3).

Multiple Sequences Alignment
Nine (9) COI gene nucleotide sequences of coccinellid beetles were aligned. Non-conserved (mutated) portions are presented by letter and identical or conserved regions were indicated by dot (Fig. 1).

Pairwise distance
The number of base differences per site from between sequences is shown. The analysis involved 9 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 353 positions in the final dataset (Table 4). Genetic distance denotes the mutation among individuals. The lowest genetic distance (0.15) was found in Hi. convergens and highest genetic distance (0.24) was found in H. axyridis.   (Fig. 2).

Fig. 1:
Multiple sequence alignment of nine predatory Coleopteran beetles. Dots denote the conserved region and color means the 60% conserved portion among these nine nucleotide sequences.  (-1850.9690).Codon positions included were 1st+2nd+3rd+Noncoding. All positions with less than 95% site coverage were eliminated. Fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. There were a total of 353 positions in the final dataset.
The bar at the bottom of the Fig. 2 and 3 provides a scale for the genetic change. In this case the line segment with the number '0.02' shows the length of branch that signifies an amount genetic change of 0.02.

Discussion
The high incidence of Black Bean Aphid might due to succulent nature of the plant (Zelena et al., 2004). Certain wasps, lady beetles (both larvae and adults), and larvae of lacewings and syrphid flies prey on aphids (Anonymous. 2000).
In the present study, the maximum predation 102±1.83 was observed by 4 th instar larvae of M. discolor followed by the 1 st instar, 2 nd instar and 3 rd instar during the year of study. The total and per day consumption of the aphids by the different instars of this grub was found important.
We observed nine coccinellid beetle found to predate the aphid population.  (Turanlı and Yoldaş, 2002;Yoldaş, 1994;Yoldaş and Sanjrani ,1999). C. septempunctata is a wellknown predator of black bean aphid (Hodek et al., 1965). A. bipunctata and C. septempunctata have been used experimentally in Finland for aphid control on chrysanthemums and roses (Mahr et al., 2010). Asian lady beetle H. axyridis is used for protecting vegetable and green crops from aphids in greenhouses of the Primorskii krai and northwestern region of the Russian Federation (Kozlova, 2008).
For accurate identification of various taxa, DNA barcoding is most promising approaches that uses molecular instead of morphological data (Blaxter, 2003). The advantage of molecular approach in determining phylogenetic relationships over the more classical approaches is that the differences can be established readily. To understand the close relationship among organisms, sequence alignment is very important method in bioinformatics for visualizing the relation (Kashmeera and Shudhikumar, 2015).
Nine coccinellid predator's COI gene sequences were collected from NCBI. The purpose of the study was to evaluate the phylogenetic relationship among coccinellid species. Among the studied sequences, the lowest nucleotide sequences were found in Sticholotis sp (440) and the highest nucleotide sequences were found in C. transversalis (683). A. bipunctata, C. septempunctata, Cy. munda, H. axyridis, S. nebulosus showed comparatively high conserved region than C. transversalis, M. discolor and Hi. convergens and Sticholotis sp in COI gene sequences.60% conserved region were showed in color region (Fig. 1).
The interspecific genetic distances for the nine species included in this study had range from 0.15 to 0.24.Comparatively higher interspecific genetic diversity recorded for H. axyridis (Table 4).
In Phylogenetic analysis, the constructions of trees were done by both Neighbor joining (NJ) and Maximum  Fig. 2 and Fig. 3).
Very limited molecular experiments have been stated on Coccinellidae, matched to other insect groups in the world. Fu and Zhang (2006) was the pioneer of molecular systematic analyses of family Coccinellidae. Kobayashi et al. (1998) studied Cytochrome Oxidase I (COI) gene region of Epilachninae. Von der Schulenburg et al. (2011) also studied on coccinellidae and concluded that extreme lengths and length variation of Internal Transcribed Spacer Region I gene region has impact of species divergence. Phylogenetic analysis is important because it enriches our understanding of how genes, genome, species evolve. It also enables us to predict how they will change in upcoming generation.
Ainsley et al. (2011) conducted molecular analysis in beetle family and improved phylogenetic resolution and support for early diverging lineages. They properly distinguish the subfamilies Microweisinae and Coccinellidae. Bernhard et al. analyzed coccinellid phylogeny according to COI gene sequence. They found monophyly of coccinellidae family. The phylogenetic relationships of the three major species groups of Coleopteran were inferred using the simultaneous analysis of 642 bp of the most conserved part of mitochondrial DNA (mt DNA) cytochrome oxidase I (COI) (Bernhard et al., 2009).

Conclusions
Coccinellid beetles showed high predation efficiency especially M. discolor was found effective predator of bean aphid (Aphis fabae), which could be recommended to use in the field level after more screening. The phylogenetic analysis has led to confirm that evolution has been occurred from a common ancestor and the species are closely related to each other. That means our studied other beetles also could be recommended as effective predator of bean aphid but need further experiment to confirm this. Phylogenetic analysis and multiple sequence alignment both support the relationship of Coccinellid beetles. Proper identification of ladybird beetle would be effective for aphid control program in agriculture.