Collection and Maintenance of Termites

Reticulitermes flavipes and R. virginicus were collected in mixed forests in Washington county, and in loblolly pine (Pinus taeda L.) forests in Pearl River and Harrison counties in Mississippi. They were kept in cylindrical plastic containers (15.5 cm diameter, 4 cm deep) with 1-2 cm deep vermiculite and sand (1:1 by volume). Corrugated cardboard and/or pine wood blocks were added as food. One hundred to 2,000 individuals were collected from each termite colony. At least 15 colonies of each species were collected from each of the three counties during the period of October, 1998-August, 2000. Samples from ten C. formosanus colonies were collected from pine wood bait buried near trees in a university campus in the city of Guangzhou, China, in August 1999. One sample of C. formosanus colony was collected from a pine stump in the city of Cenxi, Guangxi, China. Two C. formosanus samples were collected from the cardboard bait buried in a city park in New Orleans, Louisiana on 15 June 1999 and 17 August 1999, respectively. Each C. formosanus sample had over 2,000 individuals. The termite colonies were kept for a maximum period of 120 d from field collection date at room temperature (21-25°C) in the laboratory.

Biology, Abundance, and Distribution of Mites and Nematodes Associated with Termites

Termites (number varied depending on purpose of the observation) from each colony were checked for mites and nematodes under a dissecting microscope (70-400×). To examine the density of Laelaptonyssus n.sp. (Laelaptonyssidae), 300 termites (workers and soldiers) were examined from each of the 3 colonies sampled from Washington County (total n = 900). The Termites were kept in the laboratory up to 30 d before examination. We assumed that the mites and nematodes associated with termites remained on/inside the termite body after the termites were transferred to the laboratory. General collections of mites were made by putting termites in 70% ethanol and immediately examining the location and number of mites. The mites dislodged slowly from termite body once submerged in 70% ethanol. To check for nematodes, termites were dissected with two No. 3 insect pins and the specimens washed with a drop of water so the nematodes could be seen in suspension. White traps were used to extract adult nematodes from termites (Kaya & Stock 1997). Ten white traps were made. Each trap consisted a 90 × 15 cm petri dish and a 5 cm diameter moist filter paper disk resting on a platform. In each white trap, 10 R. flavipes (from Washington Co.) freshly killed by fine point forceps were put on the filter paper. The traps were placed in a moist large plastic container and then maintained at 25°C in an incubator. After 10 days, tap water was added to the petri dish and the existence of nematodes was examined.

The temporal changes in abundance of mites and nematodes were examined from one laboratory reared R. flavipes colony. The colony (about 5,000 individuals) was collected in a forest in Washington Co., Mississippi and maintained in a plastic box (31 × 24 × 11 cm) with 5 cm deep sand and pieces of corrugated cardboard. Twenty to 120 workers were examined weekly. We also checked weekly for presence of adult nematodes in the rearing medium and around dead termites using a dissecting microscope.

To examine the relationship between the abundance of mites and nematodes, the same individuals of R. flavipes and R. virginicus were examined for both mites and nematodes. They were first checked using a dissecting microscope for mite densities, then they were put on a glass slide and dissected to check for presence of nematodes. Nematodes and mites from C. formosanus were examined from different individuals. Termites had been kept in the laboratory for 60-80 d at examination date since collection.

Different parts of the termite body, e.g., head, thorax, and abdomen, were examined separately for nematodes. Termites from three R. flavipes colonies (total of 39 termites) and four R. virginicus colonies (total of 85 termites) were examined. Voucher specimens have been deposited in collections of the Stoneville Research Quarantine Facility, USDA Agricultural Research Service, Stoneville, MS. and Museum of Zoology, Michigan State University, Ann Arbor, Michigan.

Effect of Australhypopus sp. on R. flavipes

The most abundant mite in Reticulitermes colonies was an undescribed species in the genus Australhypopus (Acaridae). The only species currently placed in this genus is the type-species, A. flagellifer Fain and Friend, described from deutonymphs collected from feces of a numbat, Myrmecobius fasciatus Waterhouse, a termitophagous marsupial, in Western Australia (Fain & Friend 1984). Examination of type material by B. O’Connor indicates that three other termitophilous acarid species should also be placed in this genus: Acotyledon formosani Phillipsen and Coppel, A. lishimei Samsinak, and Tyroglyphus viduus Berlese and Leonardi. A systematic revision of this genus is in preparation by B. O’Connor and A. Huang (University of Michigan).

In our laboratory colonies of both species of Reticulitermes, when dead termites were present there were enormous numbers of Australhypopus sp. This mite was propagated by mixing dead termites (killed by freezing) with healthy termites. The termites were then placed in cylindrical plastic containers (15.5 cm diameter, 4 cm deep) with 1 cm deep vermiculite and sand as rearing medium and corrugated cardboard as food. Termites were kept at room temperature (21-25°C). Large numbers of adults and nymphs of Australhypopus sp. were present 15 d later and were harvested by brushing them from the inside surface of the container into a small round plastic container (5.0 cm diameter, 3.5 cm deep) filled with 0.01% Triton X-100 (wetting agent) (Sigma-Aldrich, Inc.) fluid. Mite density was determined. Mites (in feeding stages) were then transferred to 100 × 15 mm petri dishes with 40 healthy termite workers per dish and a corrugated cardboard disk at rate of 5 and 10 mites per worker (200 and 400 mites per dish, respectively) using a 5 ml pipet. A total of 5 ml solution was added to each dish. The mites evenly dispersed soon after being transferred to the dishes. The experiment was a completely random design with each treatment rate was replicated three times. The control dishes received only 0.01% Triton X-100 solution. All of the replicates were from the same termite colony. Dishes were kept at 26°C, 85% RH in a dark chamber. Observations for termite mortality were made every 7 d for 5 wk.

Statistical Analysis

Mortality data were arcsine of the square root transformed and analyzed using repeated measures Analysis of Variance (ANOVA) (Littell et al. 1996). Analysis was performed using PROC MIXED in SAS software (SAS Institute 1999).

Biology, Distribution, and Abundance of Mites Associated with Termites

Examination and dissection of R. flavipes, R. virginicus, and C. formosanus colonies showed that the mite fauna associated with the three termite species was quite diverse (Table 2). The mites were found mostly on the termite heads, although some were also found on the thoraces, abdomens, and legs.

Australhypopus sp. (Acaridae), the most common mite species found on both R. flavipes and R. virginicus, has a distinct phoretic stage, the deutonymph (termed “hypopus” in older literature), in addition to the trophic stages. The deutonymphs were most often seen attaching to termite heads and mouthparts. They do not have feeding mouthparts, and therefore cannot feed on termites. Once the termites die from injury or other causes such as disease, deutonymphs soon (as early as 24 h after host death) transform into tritonymphs, one of the trophic stages, and feed on the dead termite body. Adult Australhypopus sp. emerged as early as 1 d after emergence of tritonymphs. Adults lay eggs near dead termites. The eggs were laid singly or in batches of 2 to 30. The eggs hatched after 1-3 d. High mite densities were found in weak termite colonies, which were characterized by individuals showing lower body weight and flatter abdomens.

Enormous numbers of Australhypopus sp. deutonymphs were found on the inside wall of the containers holding R. flavipes colonies in the laboratory. The head and legs of R. flavipes workers were found covered by deutonymphs of Australhypopus sp. Thousands of dead Australhypopus sp. deutonymphs also could be found immediately outside of the termite rearing containers. Two factors were found closely related with high mite populations: the existence of injured or dead termites; and very low moisture content in the rearing medium.

Schwiebea sp. and Histiostoma sp. also were commonly seen on R. flavipes and R. virginicus, but did not occur as often as Australhypopus sp. Laelaptonyssus n.sp. (Laelaptonyssidae) (Krantz 2001) was found in 5 of the R. flavipes colonies of the 30-40 colonies examined. This is a rare species and apparently lives only on termites. Numbers were always low. Among three R. flavipes colonies that had this mite, the mite density ranged from 1.3 to 17.3 per hundred termites (mean = 9.2%, n = 900). Up to three Laelaptonyssus n.sp. were seen on one termite. They were mostly seen on the dorsal or lateral sides of the termite abdomen, and moved rapidly when disturbed. This mite died soon after the termite host died.

On C. formosanus, the dominant mite species were Histiostoma formosana Phillipsen and Coppel and Cosmoglyphus absoloni (Samsinak). Histiostoma formosana deutonymphs were phoretic and were mostly found on termite tarsi and tibia, whereas deutonymphs of C. absoloni normally attached to the termite head. When H. formosana populations were high (≥15 per termite), they could be seen on the whole body. These two species appeared in great numbers (∃20 per termite) in weak, laboratory-reared colonies. Histiostoma sp., Australhypopus sp., and Schwiebea sp. also were common in C. formosanus colonies held in the laboratory. Laelaptonyssus chinensis Samsinak, a rarely observed species that lives only on termites, was found in one colony collected from Cenxi, Guangxi, China.

Distribution and Abundance of Nematodes in Termites

Nematodes were often seen inside the termite body. The only nematode observed was Rhabditis sp. (Rhabditida: Rhabditidae). Among the three termite species, nematodes were most common in R. flavipes and least common in C. formosanus. The maximum number of nematodes per termite also was different (Table 3). We did not find the adult form of the nematodes either by observation of the termites externally or by extracting nematodes from termites using the white trap method.

The distribution of nematodes in three body parts was similar in R. flavipes and R. virginicus (Fig. 1). Most of the nematodes were found in termite heads with an average of 85.8% of the total occurrences in R. flavipes and R. virginicus. Only 7.0% and 7.2% of the nematodes were found in the thorax and abdomen, respectively. Due to the low count, distribution of nematodes in different C. formosanus body parts could not be described.

Temporal Changes in Mite and Nematode Populations

Mite and nematode populations varied not only between termite colonies, but also changed over time. One R. flavipes colony (about 5,000 individuals) was observed in the laboratory for three months (Fig. 2). The density of mites was 1.1 (n = 47) per termite when termites were collected from the field. The mite densities dropped and fluctuated to 0.1-0.3 mites per termite when held in the laboratory. The percentage of termites infested with mites varied between 10 and 32.2% (n = 20 to 120). Average nematode numbers per termite also fluctuated between 0.8 and 2.5. The percentages of termites with nematodes varied between 60-100% (n = 20) from 4 examination dates.

The C. formosanus colonies collected from China showed a dramatic increase in mite population and decrease in body weight and termite numbers. Five colonies were observed for their mite populations and body weight. The weight of C. formosanus (4% soldiers) was measured as 3.58 ± 0.01 mg/termite (279 ± 0.6 termites/g) at collection date. The average mite density was 1.0 mite/termite 70 d after being kept in laboratory. After 111 d of rearing in laboratory, the mite density increased to 24.4 mites/termite. Most of the mites belonged to Histiostoma spp. The average weight of termites decreased to 0.14 mg/termite (7,407 termites/g).

High mite density could be lowered if termites are kept in appropriate conditions. We observed a colony of R. flavipes (with over 5,000 individuals) maintained in a 10-gallon aquarium (51 × 25.5 × 30.5 cm). The container was filled with 2 cm deep vermiculite and 8 cm deep of southern pine wooden stakes. The inside walls of the aquarium were covered with enormous numbers of Australhypopus sp. deutonymphs 2-3 wk after termites were transferred from the field. This was caused by the existence of injured termites. Mites disappeared from the inner walls of the container later. This suggests that there was a cycle of reproduction of mites on the dead termites, followed by a population crash from the lack of dead termites.

Relationship between Mites and Termites

Although a high density of Australhypopus sp. was observed on R. flavipes and R. virginicus, it was not observed to cause significant mortality to termites in the laboratory. The adults and tritonymphs were observed feeding only on dead termites. Termite mortality was not significantly higher when mites were added to the termite colony (F = 1.53; df = 2, 6; P = 0.29) (Fig. 3). Termite individuals with high numbers of mites (10 mites per termite) were capable of living for many weeks. Therefore, the presence of large numbers of Australhypopus sp. did not appear to cause significant health problems for termite colonies. The mite species C. absoloni from termite colonies collected in China showed a similar relationship with C. formosanus.

Relationship between Rhabditis sp. and Termites

The presence of nematodes in the three termite species did not cause any abnormal morphology or behavior of the termites. We often observed nematodes attached to the mouthparts of healthy R. flavipes and R. virginicus when they were immersed in 70% ethyl alcohol. This demonstrates that the nematodes were located close to the immediate opening of termites’ mouthparts. Dead R. flavipes, R. virginicus, and C. formosanus placed in rearing containers and on white traps did not yield nematodes. No nematodes were found in dead termites when dissected in water.