Dear all,

I recently posted a question with regards insect/lepidopteran
housekeeping primers as a control for wolbachia assays. My original
email was as follows:

> Can anyone recommend a primer set for a general insect or
> Lepidopteran-specific housekeeping gene? I have been trying to amplify CO1
> (see Lunt et al 1996, Ins. Mol. Biol. 5: 153) as a control for Wolbachia
> assays, but its not working as well as I would like, so I wish to give
> something else a try.
>

Many many thanks to everyone that gave me advice - in case anyone else
is interested, some of the answers i received are copied below.

Cheers,

Zen

--
Dr Zenobia Lewis

Post-doctoral Research Fellow
Laboratory of Evolutionary Ecology
Graduate School of Environmental Science
Okayama University
Japan

In our lab, we work on bumblebees and use RPS5 (ribosomal protein S5)
as our housekeeping gene,
though we do have primers also for actin. RPS5 seems to work very well, and
was initially used by Evans (2004) Journal of Invertebrate Pathology 85
(2004) 105­111 for immune studies. Our housekeeping primers were re-designed
specifically for bumblebees, but it might be worth a go using Evans's
honeybee primers, as RPS5 is very well conserved between bees, butterflies
nd wasps (>70% nucleotide homologay).

You might want to check out EF1-alpha.  The Lep primers won't amplify
the EF1-alpha from Wolbachia.  For primers and conditions, see:
Monteiro and Pierce.  2001.  Mol. Phylogene. Evol. 18:  264-281.

I have had excellent luck with COI primers 1709 and 2353, in a wide
variety of Lepidoptera as well as other insects. The reference for those
primers is:
Simon, C., T. R. Buckley, F. Frati, J. B. Stewart, and A. T. Beckenbach.
2006. Incorporating molecular evolution into phylogenetic analysis, and a
new compilation of conserved polymerase chain reaction primers for animal
mitochondrial DNA. Annual Review of Ecology, Evolution, and Systematics
37:545-579.

I've been running the Jerry/Pat2 primer pair (Simon et al. 1994 but
the Pat is modified in Murray and Pashley Prowell 2004) in my
Wolbachia assays.

I'm working on wasps, but some of the primers I use should be general
enough to work for Lepidoptera as well. You might want to check Brian
Danforth's page, which gives an excellent overview of primers used
primarily in bees, but it's also the best one I know for insects in
general:
http://www.entomology.cornell.edu/BeePhylogeny/index.html

We have successfully sequenced wingless, EF1-alpha, Arginine kinase and
LW rhodopsin with the primers given there.
Otherwise, you could try beta-actin, which is often used as a
housekeeping gene control (if you need it for quantification by qPCR,
that's probably the gene of choice). For our wasps, we designed some
primers, and Actin-F1 and -R1 worked quite well (over a range of
annealing temperatures, from 45-56 C):
Actin-F1  5'-GGTAACGAAAGATTCCGTTG-3'
Actin-R1  5'-GATCCACATCTGTTGGAAGG-3'
They amplify a fragment of about 320 bp of beta-actin.
I don't know whether they would work for Lepidoptera, you should check
some Lepidopteran actin sequences from GenBank whether the primers
match.

In the honeybee Apis mellifera, actin has been used, and also Krh I and Rp49
(Navajas et al 2008, BMC Genomics), or ribosomal protein S5 (Schlüns &
Crozier 2007, Ins Mol Biol) etc. Maybe they can also be used for other
insects?

 Try the primers in :
Evolutionary relationships of Drosophila mojavensis geographic host
races and their sister species Drosophila arizonae
L. K. REED, M. NYBOER, T. A. MARKOW
Molecular Ecology
Volume 16, Issue 5 , Pages1007 - 1022
I had a lot of luck with these universal CO1 primers.

zen.lewis@googlemail.com