Molecular characterization of mitochondrial COI gene sequences in Micraspis allardi from Pakistan.

The Coccinellidae is a highly diversified family of order Coleoptera. Coccinellid ladybirds are well known for their role as biological control agent against varied range of agricultural pests. The samples of coccinellid ladybird collected from Pakistan were identified and characterized as Micraspis allardi (Mulsant, 1866). This is one of the least-studied ladybird species with limited work on its ecological distribution as a biological control agent. The genus Micraspis has vast genetic diversity with a possible presence of unknown number of cryptic species. Sequence information of some species of the genus Micraspis are present in NCBI database. However, least molecular data or sequences describing M. allardi could be available from database. Therefore, morphological and molecular characterization was imperative for this species. Here, the samples collected from sugarcane field of Faisalabad District of Pakistan and were identified by using morphological and molecular protocols. For molecular identification, two different regions of mitochondrial cytochrome c oxidase I (COI) gene (COI-5' and COI- 3') were used as molecular markers for the identification of the species. Morphological appearance, DNA sequence similarity searches and phylogenetic analysis collectively indicated it as M. allardi. To the best of our knowledge, this is the first report providing molecular evidence of M. allardi using mitochondrial DNA barcode region (658bp) as well as mtCOI-3' sequences (817bp). The study will help in understanding population genetics through diversity analysis, ecological role, and phenotypic structures associated with the geographic range of this species.

A DNA barcode survey of insect biodiversity in Pakistan.

Although Pakistan has rich biodiversity, many groups are poorly known, particularly insects. To address this gap, we employed DNA barcoding to survey its insect diversity. Specimens obtained through diverse collecting methods at 1,858 sites across Pakistan from 2010-2019 were examined for sequence variation in the 658 bp barcode region of the cytochrome c oxidase 1 (COI) gene. Sequences from nearly 49,000 specimens were assigned to 6,590 Barcode Index Numbers (BINs), a proxy for species, and most (88%) also possessed a representative image on the Barcode of Life Data System (BOLD). By coupling morphological inspections with barcode matches on BOLD, every BIN was assigned to an order (19) and most (99.8%) were placed to a family (362). However, just 40% of the BINs were assigned to a genus (1,375) and 21% to a species (1,364). Five orders (Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera) accounted for 92% of the specimens and BINs. More than half of the BINs (59%) are so far only known from Pakistan, but others have also been reported from Bangladesh (13%), India (12%), and China (8%). Representing the first DNA barcode survey of the insect fauna in any South Asian country, this study provides the foundation for a complete inventory of the insect fauna in Pakistan while also contributing to the global DNA barcode reference library.

Transmission of cotton leaf curl disease: answer to a long-standing question.

Cotton leaf curl disease (CLCuD) has been a major constraint to cotton production across Pakistan and northwestern India since the early 1990s. The disease is caused by a number of begomoviruses, including Cotton leaf curl Multan virus (CLCuMuV), which associate with a specific host range and symptom determining betasatellite known as Cotton leaf curl Multan betasatellite (CLCuMuB). Bemisia tabaci is a complex of cryptic species that consists of numerous (> 44) morphologically indistinguishable and, at least partially, reproductively isolated species. CLCuD has recently been introduced into parts of China but has not, at least so far, become a problem in the major cotton regions. The disease in China has been shown to be caused by CLCuMuV with CLCuMuB, which is believed to have been introduced from South Asia in ornamental plants. To understand the basis for this lack of spread of CLCuD into the cotton-growing areas of China, Pan et al. (Phytopathology 108:1172-1183, 2018) investigated the transmission of CLCuMuV/CLCuMuB by B. tabaci. The study showed that, of the four cryptic B. tabaci species investigated, only the cryptic species Asia II 1 was able to efficiently transmit CLCuMuV/CLCuMuB. Significantly, Asia II 1 is not present in the major cotton-growing regions of China. The results of Pan et al. (Phytopathology 108:1172-1183, 2018) are discussed with particular emphasis on the situation of CLCuD in Pakistan and northwestern India, which differs significantly from the situation in China.

Diversity and Distribution of Cryptic Species of the Bemisia tabaci (Hemiptera: Aleyrodidae) complex in Pakistan.

Bemisia tabaci (Gennadius; Hempitera: Aleyrodidae) is considered to be a cryptic (sibling) species complex, the members of which exhibit morphological invariability while being genetically and behaviorally distinct. Members of the complex are agricultural pests that cause direct damage by feeding on plants, and indirectly by transmitting viruses that cause diseases leading to reduced crop yield and quality. In Pakistan, cotton leaf curl disease, caused by multiple begomovirus species, is the most economically important viral disease of cotton. In the study outlined here, the diversity and geographic distribution of B. tabaci cryptic species was investigated by analyzing a taxonomically informative fragment of the mitochondrial cytochrome c oxidase 1 gene (mtCOI-3'). The mtCOI-3' sequence was determined for 285 adult whiteflies and found to represent six cryptic species, the most numerous being Asia II-1 and Middle East Asia Minor 1 (MEAM-1), the later also referred to as the B-biotype, which was previously thought to be confined to Sindh province but herein, was also found to be present in the Punjab province. The endemic Asia I was restricted to Sindh province, while an individual in the Asia II-8 was identified in Pakistan for the first time. Also for the first time, samples were collected from northwestern Pakistan and Asia II-1 was identified. Results indicate that in Pakistan the overall diversity of B. tabaci cryptic species is high and, based on comparisons with findings from previous studies, the distribution is dynamic.

High diversity and rapid diversification in the head louse, Pediculus humanus (Pediculidae: Phthiraptera).

The study analyzes sequence variation of two mitochondrial genes (COI, cytb) in Pediculus humanus from three countries (Egypt, Pakistan, South Africa) that have received little prior attention, and integrates these results with prior data. Analysis indicates a maximum K2P distance of 10.3% among 960 COI sequences and 13.8% among 479 cytb sequences. Three analytical methods (BIN, PTP, ABGD) reveal five concordant OTUs for COI and cytb. Neighbor-Joining analysis of the COI sequences confirm five clusters; three corresponding to previously recognized mitochondrial clades A, B, C and two new clades, "D" and "E", showing 2.3% and 2.8% divergence from their nearest neighbors (NN). Cytb data corroborate five clusters showing that clades "D" and "E" are both 4.6% divergent from their respective NN clades. Phylogenetic analysis supports the monophyly of all clusters recovered by NJ analysis. Divergence time estimates suggest that the earliest split of P. humanus clades occurred slightly more than one million years ago (MYa) and the latest about 0.3 MYa. Sequence divergences in COI and cytb among the five clades of P. humanus are 10X those in their human host, a difference that likely reflects both rate acceleration and the acquisition of lice clades from several archaic hominid lineages.