Publications from the Mississippi State University component of RMMP were mostly in "grey literature". Eventually, we hoped to publish our results in major journals, but because I left for the UK and Bob Fritzius also left MSU soon afterwards, publication of our data in peer-reviewed journals was never achieved.

Nonetheless, the results of our work may be of interest to others in the fields of Anopheles systematics, mosquito control and public health, and insecticide resistance. I am therefore making these reports available here as fax-quality PDF files, and some of the data as text files.

A. Genetic differences between members of the Anopheles quadrimaculatus species group

In Mississippi, three sibling species were identified on the basis of allozyme polymorphisms (Mallet 1989); we used electrophoretic techniques originally developed by Greg Lanzaro, and refined by Sudhir Narang, Paul Kaiser & Jack Seawright and others at the USDA Gainesville laboratory to identify the species.  An excellent review of their biology and taxonomy has been produced by Reinert et al. (1997), together with a complete list of references to all the available literature.

We used the letter species designations current at the time in this group to delimit species. In 1989, we used allozyme work to discover what we thought was new species we called "C?" in Noxubee Wildlife Refuge in 1989; this turned out to be "species D" (i.e. An. maverlius); the other two found in Mississippi were "species A" and "species B".   Eventually detailed studies of morphological differences were done, and the species were named as follows:
 

"species A"	Anopheles quadrimaculatus Say 1824
"species B"	Anopheles smaragdinus Reinert 1997
"species C" or "C1"	Anopheles diluvialis Reinert 1997
"species C2"	Anopheles inundatus Reinert 1997
"species D" ("C?" of Mallet 1989)	Anopheles maverlius Reinert 1997

B. Larval microhabitat differences between species of Anopheles in Noxubee Wildlife Refuge, Mississippi

We made a detailed examination of Anopheles larvae across the water bodies in Noxubee Wildlife Refuge, Noxubee County near Starkville, Mississippi.  Brief conclusions were: (1) Anopheles punctipennis is commoner in moving than in standing water, in shady sites than in sunny sites, and later in the season than earlier (Mallet 1991).   Anopheles punctipennis also occurred early in the season in small roadside puddles (which perhaps had been connected in small rivulets during rainy weather; Mallet 1990).  (2) Anopheles crucians is commoner in sunny sites in July and August than at any other time or place.  It differs little in its habitat preference to the Anopheles quadrimaculatus species group (Mallet 1991).  (3) The Anopheles quadrimaculatus group species are commoner in still water in July and August than at other times (Mallet 1991).  (4) The different species within the Anopheles quadrimaculatus group do not show any clear differences in larval microhabitat (Mallet 1991), although there are slight significant differences of frequency of the three species in adult roosting collections in Noxubee (Mallet 1990).

C. Estimate of the rate of hybridization, and species concepts

We obtained mated females from adult roosts in mixed populations of An. quadrimaculatus, An. smaragdinus and An. maverlius in Noxubee Wildlife Refuge.  These were induced to lay eggs in small vials.  The offspring, consisting of many hundreds of broods of the three species, were analysed using allozymes.  The broods were always of the same type as the female, and never showed evidence of being hybrids.  If natural hybridization occurred, it was extremely rare, and we did not detect any.  The rate of hybridization seems to be zero!

It may be of interest to note that I was initially skeptical of the differences between the species and suspected there might have been some electrophoretic artifacts involved in their separation.  (I was also interested in the possibility of hybridization, which occurs between many other sibling species). As it turned out, the differentiated allozyme loci in our three species mendelized extremely well, and the genetic differences were real.

The fact that we could tell species apart solely on the basis of gene frequency differences was a formative event for me, and indeed was one of the data sets leading to my proposal that species are readily identified as "genotypic clusters" (Mallet 1995).  That paper also cited Bob Fritzius' and my work on Anopheles. Today, perhaps, "genomic clusters" would be a better term.

D. Distribution of Anopheles across the state of Mississippi, and the effect of gross habitat

We also carried out a survey of quadrimaculatus group species across the state of Mississippi in 1990 (Fritzius 2002a), 1992 (Fritzius 2002b), and 1993 (Fritzius 2003) and found strong differences in their distributions.  An. quadrimaculatus is the commonest, and is virtually the only species found in the Mississippi Delta, where it is a major biting pest.  An. smaragdinus and An. maverlius are much more sporadic in their distributions and are generally restricted to woods away from  human habitation (Mallet 1990, 1991), perhaps because of their susceptibility to insecticides (see E below).

E. Insecticide resistance

We tested adult Anopheles quadrimaculatus group species for insecticide tolerance to insecticides using a vial test invented by F.W. Plapp for rapid sampling of Heliothis virescens (tobacco budworm).  The test has the advantage that individuals are tested separately; overcoming the non-independence of multiple individual testing in the WHO tests. To ensure the correct species, we tested many broods reared from adult mated females, induced to lay as above (C), and for which we identified some members of the brood using allozymes.  We compared our results to the "Florida USDA" strain of An. quadrimaculatus sensu stricto that had been in captivity since the 1940s, long before widespread use of insecticide. The results can be summarized as follows: (1) An. smaragdinus and An. maverlius from Noxubee Wildlife Refuge are as susceptible to malathion and permethrin as the Florida lab strain of An. quadrimaculatus.  (2) Wild An. quadrimaculatus sensu stricto, whether from the Mississippi Delta or Noxubee, is highly resistant to malathion (about 10x - 1000x); in fact it was hard to estimate LD50s to malathion in this species since some individuals almost always survived our highest doses.  (3) Delta populations of A. quadrimaculatus sensu stricto are about 10x more tolerant of permethrin than Noxubee populations of An. quadrimaculatus sensu stricto (Mallet 1990, 1991, Mallet & Fritzius 1993).

This work suggests that malathion, widely used in mosquito control in Mississippi, might be a very poor means of control of the main pest mosquito, An. quadrimaculatus.  If Anopheles smaragdinus and An. maverlius were the pest species, then malathion might be useful; but An. quadrimaculatus is the usual pest in Mississippi (Mallet & Fritzius 1993).

The work also suggests that resistance to pyrethroids was evolving in An. quadrimaculatus to about 10x in Mississippi Delta populations near Cleveland, Mississippi (Mallet & Fritzius 1993).

References

Fritzius R. 2002a.  Data file for Anopheles quadrimaculatus group species collected across Mississippi in 1990, identified using allozymes.

Fritzius R. 2002b.  Data file for Anopheles quadrimaculatus group species collected across Mississippi in 1992, identified using allozymes.

Fritzius R. 2003.  Data file for Anopheles quadrimaculatus group species collected across Mississippi in 1992, identified using allozymes.

Mallet J. 1989. Annual Project Summary Report for Riceland Mosquito Management Program.  1 January - 31 December 1989.  USDA-CSRS Internal Document.

Mallet J. 1990. Annual Project Summary Report for Riceland Mosquito Management Program.  1 January - 30 November 1990.  USDA-CSRS Internal Document.

Mallet J. 1991. Annual Project Summary Report for Riceland Mosquito Management Program.  1 January - 30 November 1991.  USDA-CSRS Internal Document.

Mallet J & Fritzius R. 1993.  Genetic evidence for insecticide resistance in sibling species of the mosquito Anopheles quadrimaculatus.  Resistant Pest Management 5(1): 25-26.

Mallet J. 1995. A species definition for the modern synthesis. Trends in Ecology and Evolution 10: 294-299.

Reinert JF, Kaiser PE, Seawright JA. 1997. Analysis of the Anopheles (Anopheles) quadrimaculatus complex of sibling species (Diptera: Culicidae) using morphological, cytological, molecular, genetic, biochemical, and ecological techniques in an integrated approach. Journal of the American Mosquito Control Association, vol. 13 (Supplement), 1-102. [NOTE: HUGE ACROBAT FILE!! Approx. 10MB]