Megacopta cribraria ( F.) 311
5.5.2 Nuclear Markers
The 18S rRNA and EF1-α nuclear gene fragments (Table 5.2) also were amplified and sequenced from
randomly chosen adult bugs. As with the mitochondrial gene sequences, they also showed consensus
among all collections studied. This lack of variation among these gene and mtDNA gene sequences
strongly indicates a founding event exacerbated by a severe genetic bottleneck such as the introduction
of a single female.
5.5.3 Endosymbionts
Using primers 16SA1 and 16SB1 specific for Candidatus Ishikawaella capsulata (Hosokawa et al. 2006),
the 16S rRNA gene was amplified and sequenced (Jenkins et al. 2009) in three of the originally collected
Megacopta cribraria individuals. A GenBank Basic Local Alignment Search Tool (BLAST) search
(Altschul et al. 1990) matched for Candidatus Ishikawaella capsulata (AB067723) and all three speci-
mens contained the endosymbiont (Jenkins et al. 2010). Furthermore, all individuals randomly collected
in the United States and those intercepted in Honduras in 2012 had the same Candidatus Ishikawaella
capsulata sequence as did random samples collected in Fukuoka Province, Japan, and Gyeongnam
and Jeonnam, South Korea in 2012. Other 16S rRNA sequences from M. cribraria in China matched
more closely gram-negative β-proteobacterium, genus Achromobacter (GU086442) (Tracie M. Jenkins,
unpublished data). Brown et al. (2013) confirmed the identity of the symbiont and suggested that M.
cribraria likely arrived in the United States with the capability of being a soybean pest.
A secondary endosymbiont, the α-proteobacterium Wolbachia, also was discovered in Megacopta
cribraria collected in Georgia (Table 5.2). This endosymbiont may function to increase insect fecundity
through increased growth and development (Dobson et al. 2002, Hosokawa et al. 2007b). A 548 bp frag-
ment of the outer surface gene wsp was sequenced from M. cribraria collected in Georgia (GenBank
#JQ266093) (Jenkins and Eaton 2011). A BLAST search then revealed a consensus with the wsp gene
fragment identified as strain 1 (GenBank # AB109596), which was collected from M. cribraria (as
M. punctatissima) in Japan (Kikuchi and Fukatsu 2003). Thus, the primary and secondary endosymbi-
onts found in Georgia populations of M. cribraria were the same as those found in Japan.
5.5.4 Origin of Megacopta cribraria
Hosokawa et al. (2014) extracted DNA from 50 adult insects identified as Megacopta cribraria or
M. punctatissima from native populations in Japan, South Korea, China, and Vietnam. Additionally,
DNA was extracted from three M. cribraria adults collected in the southeastern United States. Using
the M. cribraria mitochondrial genome sequence (GenBank # JF288758) as a reference (Jenkins and
Eaton 2011), primers were designed to amplify and sequence the ND2-ND4 mtDNA region (8,687 bp),
TABLE 5.2
Sequences and GenBank Numbers for Megacopta cribraria
Gene Fragment or Genome Sequence Length (bp) GenBank Number
MtDNA Complete Genome (GA1) 15,647 JF288758
COI-L-COII (GA1-southeastern U.S) 2336 HQ444175
COI-L-COII (GA1-Honduras) 2336 KF186211-KF186226
COI-L-COII (HN1, Honduras) 2336 KF186227
COI-L-COII (Asia) 2336 KF186196-KF186210,
KC153656-KC153704
18S rRNA 1445 JQ254888
EF-1σ (Elongation Factor 1- alpha)^559 KF356283-KF356297
16S rRNA (C.I. capsulata (Cic-1) 1363 JF732916
groEL protein (C. I. capsulata (Cic-1) 1547 JF736508
wsp (outer surface protein-Wolbachia sp) 548 JQ266093