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United States Department of Agriculture

Agricultural Research Service

Saltcedar: Diorhabda elongata Field Release
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Saltcedar: Diorhabda elongata Field Release

 

Life-History of Diorhabda elongata in Secure Field Cages:
Results of Research during Stage A of Research Releases in 1999


 

Text Box: Data Provided by:CA: Raymond Carruthers (USDA-ARS), Tom Dudley (University of California, Berkeley)CO: Debra Eberts (USDI-BR)NV: Jeff Knight (State of Nevada, Division of Agriculture)TX: Jack DeLoach, Phil Lewis, James Tracy (USDA-ARS), Allen Knutson (Texas A&M University)UT: Greg Abbott (USDA-APHIS), Pat Fosse (USDI-BLM)WY: Dave Kazmer (University of Wyoming), Gary Adams, John Larson (USDA-APHIS)Overseas Cooperators:China:  Baoping Li (Xinjiang Agricultural University), Hongyin Chen (Sino-American Biological Control Laboratory)Kazakhstan:  Ivan Mityaev, Roman Jashenko (Kazakhstan Academy of Sciences)
Diorhabda elongata adult
on saltcedar foliage 

The target: saltcedar (Tamarix ramosissima

       Compiled by:

       Juli Gould

TABLE OF CONTENTS
 
Executive Summary 1
Introduction 2
Release Site Locations and Insect Releases 4
Nursery Cages 4
Field Cage Releases 4
Number of generations per year 5
Developmental Time Related to Temperature 6
Mortality Factors affecting D. elongata 8
Net Reproductive Rate 9
Damage to Saltcedar by Diorhabda elongata 10
Summer Aestivation and Over-Wintering Diapause 11
Damage from Leafhoppers 13
Monitoring Plans and Baseline Data 14
Recommendations 14
Individual Site Summaries 15
The Albany, CA Quarantine Facility 15
The Temple, TX Quarantine Facility 16
Fort Hunter Liggett, Monterery Co., California 16
Independence, California 17
Pueblo, Colorado 18
Lovelock and Schurz, Nevada 18
Seymour, Texas 19
Temple, Texas 19
Delta, Utah 21
Lovell, Wyoming 21


Executive Summary

The results reported here comply with the monitoring requirements in the first year monitoring plan for Stage A of research on the saltcedar leafbeetle, Diorhabda elongata as required by the release permits issued by USDA-APHIS-PPQ for each site. The monitoring plan was approved by the US Fish and Wildlife Service in a Letter of Concurrence issued 4 June 1999. This research was conducted in secure field cages at eight sites in six states (CA, CO, NV, TX, UT, and WY). These results are presented so that a decision can be made on proceeding with Stage B research, the release of D. elongata outside of cages at approved circumscribed sites that are more than 200 miles from areas where the endangered southwestern willow flycatcher, Empidonax traillii extimus, nests in saltcedar.
 
Diorhabda elongata were released either onto exposed branches within secure field cages or into organdy sleeves inside the cages, where development of individuals could be monitored. Data within the cages were collected on beetle development throughout the life-cycle, reproductive rate, damage to saltcedar, over-wintering/diapause, and aestivation. Because permits were not issued for release at many of the sites until mid-July, we were able to conduct research on more than one generation only at two approved nursery cage sites where beetles were released in 1998.
 
At Pueblo, CO, Diorhabda elongata over-wintered as adults that emerged onto saltcedar in the field cages in the spring, around the time that saltcedar was producing its first leaves. Eggs were laid on saltcedar foliage and hatched into larvae that developed through three instars. Third instar larvae moved to the litter or soil beneath the saltcedar where they pupated. As is typical for insects, D. elongata developed more quickly at the more southern sites where temperatures were higher. Development from egg to adult took between 26 days at Temple, TX and 49 days at Pueblo, CO. At Temple, D. elongata developed considerably faster in field cages where temperatures averaged 24.4oC (29 days) than in the laboratory at a constant 24.1oC (37 days). Significantly more beetles were found on branches in the southern and eastern directions, suggesting that D. elongata can alter its behavior to gain more heat units and develop more rapidly.

Although the beetles developed more rapidly at warmer sites, we did not see more than two generations at any location. At the warmer sites, the beetles became inactive when the temperatures became quite hot and when photoperiods began to decline. In early August, adult beetles that emerged at the end of the second generation were seen on the saltcedar foliage, but they were not seen mating and did not lay any eggs. In August and September, these adults ceased activity. Detailed research in Temple, TX indicated that the cessation of adult activity and reproduction might be influenced by photoperiod and/or temperature and could be reversed by changing these environmental conditions.

In sleeve studies at Independence, CA, only 10% of individuals survived from the egg to the adult stage. Survival from first instar to the adult stage at other sites ranged from 33% to 76%. When allowed to move freely in the cages, the D. elongata population in Pueblo, CO increased twenty-five fold from the 130 over-wintering adults to the adults produced at the end of the first generation. It should be noted, however, that organdy sleeves and field cages are artificial environments and estimates of survival and reproductive rates may not estimate population increases outside of field cages. In general, predators, parasitoids, and pathogens that could contribute to D. elongata mortality were excluded from the cages, although spiders and ants were seen preying on D. elongata at some of the sites.

The greatest mortality to D. elongata was not caused by predators but by a competitor, the leafhopper, Opsius stactogalus. This leafhopper caused the death of so much saltcedar foliage (often approaching 100% of the foliage in some cages), that D. elongata had to be moved to new cages to avoid starvation. Defoliated saltcedar did, however, re-foliate in all cases. Although leafhoppers rarely reach high density or cause considerable damage outside of field cages, they consistently caused much damage in almost all the release cages. The extremely different population densities and damage levels attributed to leafhoppers inside and outside cages further illustrates the difficulty in predicting the effect of an insect on a given plant from cage studies. It is not known whether the cages protected the leafhoppers from predators or provided a more suitable mircroclimate, but clearly their performance in the cages is not realized in the open field. The corollary, of course, is that we cannot accurately predict the effect that D. elongata will have on saltcedar until we can conduct research outside of field cages.

Diorhabda elongata did not feed upon any vegetation other than saltcedar in field cages, including cages having large coyote willow (Salix exigua) and gooddings willow (S. gooddingii) at Temple, TX.

Baseline data on the distribution and abundance of saltcedar and native flora were collected by taking aerial photographs of all of the research sites. Photographs were taken for approximately 8 km on either side of the field cages along the river corridor. These photographs will serve as references against which changes in the abundance of saltcedar and other large plants can be measured.

The data collected from the field cages during 1999 indicate that D. elongata will require at least several years to reduce even local saltcedar stands to a significant degree. Insect populations will take time to increase and saltcedar has a great capacity to re-foliate. This should provide time for re-vegetation, whether natural or manual, to proceed to provide satisfactory habitat for wildlife. Therefore, we recommend that D. elongata be released from the field cages during the spring of 2000 so that Phase B of the Research Plan can be implemented to measure dispersal of the beetles and their impact on saltcedar in nature.

Introduction

Saltcedar, Tamarix ramosissima, is a serious weed of riparian areas in the western United States. It infests over one million acres and is difficult and expensive to control, given its high regenerative capacity and high seed production. Over 10 years ago (1987), the Agricultural Research Service (ARS) at Temple, TX initiated a biological control program to release herbivorous insects from the native range of Tamarix against this weed in the United States. Extensive host specificity testing was conducted and the leaf beetle, Diorhabda elongata, from central Asia, and the mealybug, Trabutina mannipara, from Israel were considered safe for release. However, before releases were initiated, it was discovered that an endangered bird, the southwestern subspecies of the willow flycatcher (Empidonax traillii extimus), was utilizing saltcedar in parts of AZ, NM and NV as a nesting substrate. Because flycatcher populations were so low and the possibility of extinction was high, the Fish and Wildlife Service (FWS) wanted to proceed very cautiously with a biological control program against saltcedar, even though the saltcedar invasion is a likely major cause in the decline of the flycatcher and other threatened and endangered species. FWS wished to release the beetles only at sites far from where the flycatcher was utilizing saltcedar and to conduct careful research on the capacity of the beetles to damage saltcedar and to disperse.
 
ARS and the FWS agreed that the beetle, D. elongata, would initially be released in cages at ten circumscribed sites in six states (Texas, Colorado, Wyoming, Utah, Nevada, and California). Beetles were actually released in 1999 at eight of these sites. These sites are all located from 220 to 850 miles away and on different river drainages from where the endangered southwestern subspecies of the willow flycatcher is nesting in saltcedar. Intensive monitoring will be conducted for several years on the control insects themselves, on their effects produced on saltcedar, on recovery of the native vegetation, and on the effects on and recovery of wildlife populations, including endangered and threatened species. The monitoring will consist of intensive research at these ten sites for three years, and a decision on the future of the biological control program will be made based on the research results.
 
During the Research Phase, data will be collected both in cages for one year (Stage A) and in open field releases for two years (Stage B). This research will provide scientific information for making informed decisions regarding future releases into other areas and also on whether, or what kind of, mitigating actions may be needed in areas of Arizona and southern Nevada where the southwestern willow flycatcher is nesting in saltcedar.

During 1999 (Stage A of the Research Phase), research was conducted in secure field cages at eight sites. This research was designed to determine the survival, reproduction, feeding on saltcedar and non-target plants, and over-wintering and establishment of the biocontrol insects in the different climatic zones reproduced at the eight sites. During Stage B of the Research Phase, the cages will be removed after over-wintering has been confirmed (scheduled for mid to late spring of 2000). Some insects may be left inside the cages if more cage data are needed.

During Stage B of the Research Phase, the released insects will be intensively monitored to determine their rate of increase, mortality factors (attack by native predators, parasitoids, or pathogens), amount of damage and rapidity of control of saltcedar, amount of feeding, reproduction and damage (if any) to non-target plants, and rate of dispersal. The recovery of native vegetation and of wildlife after control will be monitored at the original sites for at least 10 years.

This report summarizes the data collected during Stage A of the Research Phase at the eight sites where D. elongata was released in secure field cages. Insects were not released in cages at two of the approved release sites.

Release Site Locations and Insect Releases

Nursery Cages

Outdoor “nursery cages” were approved by APHIS-PPQ at 4 locations; at Temple, TX and Pueblo, CO in 1997 and at Lovelock, NV and Dallas, TX in 1998. The purpose was to multiply the D. elongata population to have sufficient numbers to release when approval for field cages was received. Laboratory cultures on small, potted plants were difficult and very time consuming to produce. Beetles on large plants in outdoor nursery cages could be reared in large numbers very easily. The beetles placed in the nursery cages were from quarantine cultures that were free of any foreign parasites, predators and pathogens, and any future generations produced in the nursery cages would also be free of foreign organisms. The cages were 10 by 10 by 6 foot high, of 16 mesh plastic screening, that were secure and protected in the same manner as the later field cages. Life and seasonal history data was also taken in the nursery cages.

Field Cage Releases

Ten locations were approved in 1999 for field release for the study and dissemination of the D. elongata; nursery cages in Pueblo, CO and Lovelock, NV were re-classified as release cages. Releases actually occurred at only eight sites (Table 1).
 
Table 1: Location of sites where D. elongata was released in secure field cages.


 

State
Location
River System
Landowner
CA
Independence
Owens River
L.A. Department of Power and Water
CA
Hunter Liggett Military Reservation
Nacimiento Creek
Department of Defense, Forest Service
CO
Pueblo
Arkansas River
Bureau of Reclamation
NV
Lovelock
Humboldt River
Private
NV
Schurz
Walker River
Paiute Indian Nation
TX
Seymour
Wichita River
Private
UT
Delta
Sevier River
Bureau of Land Management
WY
Lovell
Big Horn River
Wyoming Fish and Game Department and National Park Service

The project was on track to release beetles at all sites in May 1999. A research proposal for the releases had been submitted to FWS at their request on 28 August 1998. A letter of concurrence was received from the Fish and Wildlife Service (FWS) on 28 December 1998 agreeing to the research proposal, and APHIS published an Environmental Assessment (EA) for the release of Diorhabda elongata in the Federal Register (Vol. 64, no. 52, Thursday March 18, 1999/Notices, pp. 13395-13396). However, FWS asked for an extension on the comment period to allow them to review new information. Various agencies met with FWS in May at Arlington, VA to discuss the EA. At this meeting, FWS announced that they would cancel the previously issued letter of concurrence and would issue another one. At this meeting, FWS also insisted on extensive, long-term monitoring of 1) the release insects and their behavior and effects, 2) vegetation recovery after control, and 3) wildlife recovery after control. The new Letter of Concurrence, signed 3 June 1999, allowed Phase I to proceed, but eliminated five sites that had been planned for the Rio Grande and Pecos River valleys of New Mexico and Texas. A Finding of No Significant Impact (FONSI) was issued by APHIS on 7 July 1999.

Permits for release of Diorhabda elongata in secure field cages were issued from 9 to 28 July 1999 for sites in CA, CO, NV, TX, UT, and WY. These included two former nursery cage sites at Pueblo and Lovelock. By mid-July, the beetles in the nursery cages in Pueblo, CO and Temple, TX had completed one generation. Because of the delay in permitting, however, we were only able to collect data on one generation at the remaining six sites. The progeny of beetles from spring shipments of D. elongata from China and Kazakhstan that had been through quarantine at Temple, TX and Albany, CA were used for the releases. In addition, the progeny of over-wintering beetles from Pueblo, CO were sent to other states and placed in field cages.

The field cages were typically 10 by 10 by 6 foot high metal frames held securely in place with guy wires. Sixteen-mesh screening was used as the cage material, with a zipper on one side. The bottom of the cage was sandwiched between two wooden boards that were then buried deep in the ground to prevent the escape of beetles under the cage. A protective fence surrounded all cages to prevent damage from livestock and the cages were placed in secluded areas. The integrity of the mesh screening was monitored every time the researchers visited the cage, and saltcedar plants outside the cages were checked for beetles. We know of no breaches in the security of the field cages.

Diorhabda elongata was released either into 0.3 m long organdy sleeves on individual saltcedar branches or released freely within the secure field cages. The development of individual beetles from egg to adult was followed in the sleeves. Data on beetle density and damage were collected from 12 branches (3 per cardinal direction) for the beetles released outside the sleeves.

Number of generations per year

No beetles over-wintered in the nursery cages in TX or NV, but 130 adult beetles emerged from their over-wintering sites in early May in the nursery cage at Pueblo, CO. Beetles were also released into nursery cages in May in Temple, TX. Because of the delay in release, we could determine the number of beetle generations per year only at the nursery cage sites, where beetles over-wintered from 1998 (Pueblo, CO) or were released in May 1999 (Temple, TX). Beetles at Pueblo and Temple laid eggs and larvae, pupae, and adults developed by mid-July. Beetles from the Pueblo site, as well as some from the Albany, CA and Temple, TX quarantine facilities, were then shipped to the other field sites in WY, NV, CA, TX, and UT between mid-July and early August. The beetles in Pueblo and Temple, as well as eggs or larvae released at the other sites, went on to have one more generation before entering a winter diapause. The first generation (F1) adults shipped from Pueblo to CA and TX in late July, all F1 adults shipped from Pueblo in August, and all second generation (F2) adults emerging from early August in Temple to September in CO, NV, and WY did not oviposit. Within a few weeks of appearing, these adult entered the leaf litter and did not resume activity in 1999. While we can state that it is likely that D. elongata will go through two generations in the United States, until we have beetles emerge naturally in the spring at a given site, we will not know if they can go through more than two generations. Although the beetles will probably develop more rapidly at southern sites, there is evidence that they cease reproduction in mid to late summer (August to September) in response to high temperatures or declining photoperiods (see Summer Aestivation and Over-Wintering Diapause below).

Developmental Time Related to Temperature

Studies of generation time were conducted in both the laboratory and the field in Texas. As is typical for insects, Diorhabda elongata developed more quickly in warmer temperatures. D. elongata took between 21 and 37 days to develop from egg to adult at 30°C and 24°C respectively (Fig. 1). D. elongata developed considerably more rapidly in the field cages when the average temperature was 24.4°C than in the laboratory at a constant 24.1°C. More beetles were found on branches on the southern and eastern exposures of the saltcedar trees in WY and TX, suggesting that the dark insects used solar radiation to warm their bodies, resulting in more rapid development.

 
Figure 1: Development of D. elongata at a constant temperature of 24.1°C in the laboratory at Temple, TX and at several fluctuating temperatures in the field at Temple and Dallas, TX.

Diorhabda elongata developed more quickly at the more southern sites in Texas than in Colorado, California, or Wyoming (Table 2).

Table 2: Development of D. elongata at several release sites from 1997-1999.
 

Release Site
Cage or Sleeve Release
Number of Days from Egg to Peak Adult Emergence
Lovell, WY
Cage
45
Pueblo, CO 1997
Cage
49
Pueblo, CO 1998: generation 1
Cage
48
Pueblo, CO 1998: generation 2
Cage
48.5
Independence, CA
Sleeves
40
Temple, TX
Sleeves
31
Temple, TX: generation 1
Cage
29
Temple, TX: generation 2
Cage
26

We were able to generate graphs of the seasonal occurrence of the successive life-stages of D. elongata at many of the sites. Because these graphs look essentially similar, with the main difference being the length of time taken for each life stage to develop, we will present only a few examples. Figure 2 shows the progression of D. elongata through two generations in field cages in Temple, TX. Note that the second-generation adults were not reproductively active and disappeared from the foliage after several weeks. Figure 3 illustrates the progression of the successive life-stages of D. elongata in sleeves in Independence, CA. Note that although the eggs were introduced simultaneously there was considerable overlap in the development of larvae, pupae, and adults.

Figure 2: Seasonal occurrence of D. elongata in a secure field cage in Temple, TX.

Figure 3: Seasonal occurrence of D. elongata in sleeves in Indepencence, CA.

Mortality Factors affecting D. elongata

In sleeve studies, we placed approximately 10 eggs or neonate larvae in each sleeve.Predators, parasitoids, and pathogens were excluded, and mortality was greatest in the egg stage in Independence, CA (Fig. 4).Little mortality occurred during the first instar, after which the number dying at each stage was almost constant.At the end of the generation, 10% of the individuals placed in the sleeve cages were still alive.Although data are not available for the egg stage, survival of first instar larvae through the adult stage was higher in Temple, TX (74%) and Dallas in the second generation (76%) than in Independence, CA (33%) or in Dallas during the first generation (42%).In separate experiments, placing 60 to 100 eggs each in large sleeves within Temple, TX nursery cages on 29 May 1999, survivorship was much higher in the egg stage.Survivorship was 84% (n=196) for eggs treated with distilled water, 80% (n=491) for eggs treated for 1 minute with 0.13% zephiran chloride, and 66% (n=399) for eggs treated for 1 minute with 10% chlorox. 
Figure 4: Survivorship of D. elongata from egg through adult in sleeves at four sites.
 
Generalist predators were seen attacking D. elongata in most of the field cages.Jumping spiders (Salticidae) and ants were the most common predators, however assassin bugs were also seen attacking adult beetles in Texas.At the Independence, CA site, some sleeve cages became infested with ants, and all the larvae were killed.Outside the sleeves, however, beetle larvae were seen dropping from the foliage when approached by ants.These larvae may have avoided predation but perhaps increased the risk of mortality from other factors.

The greatest mortality factor in the field cages was not predators but leafhoppers, which acted as competitors and killed the saltcedar foliage, causing D. elongata to starve.

Net Reproductive Rate

To calculate the net reproductive rate (Ro), one must know the number of individuals of a certain stage in two sequential generations.Since we had the largest number of beetles with two full generations at Pueblo, CO, we will report on the results from that site. At Pueblo, 130 over-wintering adult beetles emerged in May. At the end of the next generation, 3250 adults emerged. That is a 25-fold increase in one generation. We calculated an Ro of 25 females per female per generation (assuming a 50:50 sex ratio).This Ro observed in the field, is lower than the maximum mean Ro of 88.2 observed in the Temple laboratory for females reared on young foliage of T. ramossisima from Lovell, WY. It is, however, similar to the R­o of 29.9 for females reared on old foliage of T. ramossima from Lovelock, NV.

 
We cannot accurately predict the net reproductive rate of the beetle outside the field cage from data collected inside cages. Microclimate conditions (temperature and humidity) that affect D. elongata survival and reproduction are different inside of these field cages. Also, the beetles are not allowed to migrate from the saltcedar in the cage if resources are scarce (in Pueblo it is believed that some of the larvae may have starved as the foliage was damaged by D. elongata and leafhoppers).The cages also possibly excluded parasites and predators that might reduce the rate of increase of beetle populations outside the cages.When the beetles are released in the open field, we will determine more accurately the reproductive potential of Diorhabda elongata.

Damage to Saltcedar by Diorhabda elongata

D. elongata seems to damage saltcedar foliage by scraping tissue off the leaves rather than removing sections of the leaf. This causes sections of twigs beyond the damage to turn yellow (Fig. 5) and eventually dry up and fall off. D. elongata can, therefore, cause the death of more plant tissue than it actually consumes.

Figure 5: Young saltcedar foliage in May in Pueblo, CO damaged by adult D. elongata.
Note that the twig tips have drooped and the leaves have dried and are no longer green.

Diorhabda elongata consumed saltcedar foliage inside the field cages, but damage was quite low at most of the sites due to the low densities of D. elongata in 1999. Sometimes, especially later in the season, estimates of damage from D. elongata were confounded by simultaneous damage from leafhoppers, Opsius stactogalus

In Temple, TX, approximately 300 third instar larvae from China defoliated one of the saltcedar trees in one of the nursery cages. In Pueblo, CO, D. elongata has been in the cages for three years. In 1997, there was no appreciable damage from either beetles or leafhoppers, although leafhopper numbers were building inside the cages and causing higher damage inside the cages than outside.Leafhoppers also increased in number in the two cages where D. elongata had not been released in 1997.In 1998, the saltcedar in only one of the three cages was severely damaged (95%) by September, but this damage was due solely to leafhoppers. One fourth of the stems on these plants did not re-sprout in 1999.In 1999, the saltcedar foliage in all three cages showed significant damage (40%, 90%, and 98%). This damage was caused by a combination of D. elongata and leafhoppers. Leafhopper densities were low during the first generation of D. elongata, which caused 75% damage to the saltcedar in the cage. By the end of August, however, the foliage had re-grown quite well. The saltcedar in the cage with 90% damage had experienced a second year of defoliation in 1999, and it will be interesting to determine its ability to sprout in the spring of 2000. The saltcedar in Temple, TX also re-foliated by September, but by then leafhoppers had built up and were causing significant damage.

Saltcedar has a great capacity to re-sprout after damage from factors such as cutting and burning. We have evidence that it can also re-sprout after complete defoliation by insects. Further detailed studies will be necessary to discern the beetle densities and number of years of defoliation necessary to kill saltcedar plants of different age and size.

Summer Aestivation and Over-Wintering Diapause

Adult D. elongata were first observed to successfully over-winter in a Pueblo, CO nursery cage in 1999. Beetles emerging in May produced two generations during 1999; first generation adults emerged in mid to late July and second-generation adults emerged beginning September 8. First generation adults continued egg laying into August, but almost all second-generation adults failed to reproduce and disappeared within a few weeks after their emergence in early September. Second generation adults were produced by August 3 in nursery cages at Temple, TX, where eggs were released in May and June, and these adults disappeared without reproducing after approximately 2 weeks. Second generation adults were also produced in September from first generation adults shipped on July 20 from Pueblo to cages in Lovelock, NV and Lovell, WY, and these also failed to reproduce.First generation adults from Pueblo that were released at four sites at the end of July through early August (Hunter-Liggett, CA; Independence, CA; Seymour, TX; and Schurz, NV) also went into a reproductive and behavioral dormancy.These adults were observed to feed briefly on the saltcedar foliage but disappeared by mid-August. Upon closer inspection, adult beetles could be found in the soil or litter around the base of the saltcedar plants.Adults disappearing in mid to late summer (August to September) did not resume feeding or oviposition throughout the monitoring period of 1999. We propose that the insects entered a summer-winter reproductive diapause (aestivo-hibernation) due to a combination of high temperatures over 38°C and photoperiod changes. Eggs and larvae released into these same cages in late July or early August developed into adults, but these adults were not reproductively active and subsequently entered the litter for over-wintering.
More detailed research at Temple, TX suggests that the response of D. elongata to changes in temperature and photoperiod can be altered by further changes in temperature and photoperiod. Almost all second-generation adults entered reproductive dormancy at Temple around August 3 when high temperatures ranged from 37-39°C. However, adults that emerged in early September also failed to lay any eggs (Figs. 6 and 7, outdoor beetles in outdoor cages) and quickly entered the litter, despite the fact that maximum temperatures dropped to 32°C (which was lower than the maximum temperatures at peak oviposition in July of 33 to 33.5°C). Beetles laying eggs in the laboratory under conditions of continuous light at 27-29°C (Fig. 6 and 7, laboratory beetles moved to outdoor cages) continued to lay eggs for 10 days following transfer to the field in early September, indicating immediate temperature conditions were not unfavorable for oviposition at this time. Some beetles in the field that had ceased to lay eggs in early September, resumed egg laying after about a 2 week exposure to the laboratory conditions (Fig. 6 and 7, outdoor beetles moved to the laboratory). This was observed again when adults collected from the litter in a nursery cage on 10 November resumed laying eggs after 2 weeks in the laboratory. Further laboratory experiments are needed to confirm whether and in what way short or reducing photoperiods and temperature interact to influence the onset of dormancy and diapause.

Figure 6: Activity of adult D. elongata in outdoor field cages and in the laboratory at Temple, TX.

Figure 7: Egg laying by adult D. elongata in outdoor field cages and in the laboratory at Temple, TX.

Damage from Leafhoppers

The leafhopper, Opsius stactogalus, is not native to the United States, but it was accidentally introduced to the United States from the native range of Tamarix. It is one of only four non-native arthropods currently attacking saltcedar, the other species being a scale insect and two eriophyiid mite species, all of which cause only minor damage. Populations of Opsius rarely reach high densities in nature, and they do not seem to have a significant impact on the abundance or distribution of saltcedar. However, in many of our field cages, leafhoppers became so abundant and the saltcedar so adversely affected that beetles had to be moved to new cages or they would starve. This was in spite of the fact that no damage from leafhoppers could be detected on the saltcedar just outside of the cages. The exception to this was western NV, where leafhopper populations have been high for the last couple of years and may have caused some dieback of saltcedar. The vastly different population densities and damage levels seen due to leafhoppers inside and outside the cages very clearly illustrates the difficulty in predicting the effect of an insect on a given plant from cage studies. It is not known whether the cages protected the leafhoppers from predators or provided a more suitable microclimate, but clearly they are not the effective biological control agent that one might predict based on their effect in field cages.The corollary, of course, is that we cannot accurately predict the effect of D. elongata until we can conduct research outside of field cages.
 
In July at Temple, TX, leafhoppers were noticeably declining and were found to have died following infection by the entomopthoralean fungus Zoophthora radicans. This pathogen is a ubiquitous species and has been reported from many different climatic zones but is more common (and active) in warm (20 to 25°C) and moist habitats. It has been used in field trials against a number of leafhopper pest species in both the New and Old World.Leafhopper cadavers were shipped to Pueblo, CO and Seymour, TX in August and may have had an effect on leafhopper populations at the latter site.
 
A chemical method to control leafhoppers was successful in one of the cages in Temple, TX. The 10’ by 10’ by 6’ cage was covered with a 25’ by 25’ plastic tarp and secured at the ground with wooden boards. A 15-second burst of a pyrethrin aerosol (Whitmire ® Prescription Treatment 565 Plus XLO) was released within the cage and the tarp was left sealed for one hour before removal. The foliage remained toxic for 2-3 weeks, depending on temperature and rainfall, after which adult or larval beetles could be released in the cage. Use of the juvenile hormone mimic, Enstar II (Kinoprene), was not effective for control of leafhoppers.

Monitoring Plans and Baseline Data

Monitoring plans have been completed as required by the Research Proposal, the Letter of Concurrence, and the Release Permits. These plans cover monitoring of 1) D. elongata, 2) saltcedar and native vegetation, and 3) wildlife.Monitoring of Diorhabda elongata in field cages and after release from the cages will be done to determine its biology and behavior, reproductive rate and mortality factors, life and seasonal history, impact on saltcedar, and rate of dispersal. Vegetation will be monitored to determine the impact of insects on saltcedar health and abundance and the rate of recovery of the native plant community. The response of wildlife populations after control will also be monitored. Characterizing vegetation and wildlife responses will include the establishment of baseline data before D. elongata is released from the cages.
 
The Insect Monitoring Plan was followed during 1999 to obtain data on the life-history and behavior of D. elongata in the field cages. Baseline vegetation data in the form of aerial photography of present plant communities was obtained at all sites during 1999. Aerial photographs were taken of all of the vegetation approximately 8 km up and downstream from the field cages. These photographs will be scanned and digitized so that we can characterize the distributions of saltcedar and native vegetation prior to the release of biological control agents. In the fall, when these photographs were taken, saltcedar foliage is yellow-orange to orange-brown in color, and spectral analysis makes saltcedar distinguishable from other plant species. Aerial photographs will be taken periodically after the release of Diorhabda elongata to quantify the predicted decline in saltcedar and the anticipated return of native vegetation.

Recommendations

These experiments indicate, as best as can be determined in cage studies, that the leafbeetle, Diorhabda elongata, will not kill mature saltcedar plants in two years, and probably not for several more years, even if the tree is completely defoliated. Saltcedar plants re-vegetated in the fall after attack by D. elongata, and without a great reduction in plant size. In the more southern areas, the beetles became inactive after mid-summer, during which time the plants recovered. Based on these results, we do not anticipate a rapid decline in saltcedar numbers, even at the local release site, after initiation of biological control.
 
We recommend that Diorhabda elongata be released from the field cages at the ten approved sites so that research can proceed to Stage B of the research proposal, in which the rate of beetle dispersal and the effect of the beetle on saltcedar outside of the cages can be monitored.

Individual Site Summaries

The following summaries are mostly unedited statements submitted by the site coordinators briefly describing activities at each location in 1999. There are also summaries of activities at the two quarantine facilities.

The Albany, CA Quarantine Facility

(Submitted by Ray Carruthers, USDA-ARS, Albany, CA)
 
The Albany Quarantine Facility received two shipments of Diorhabda elongata from Dr. Lee Baoping. The insects were collected in Fukang, China and then sent to the US via direct airfreight from the ARS SinoAmerican Biological Control Laboratory in Beijing China.The first shipment contained approximately 1000 beetles and the second contained approximately 3500 individuals. These two shipments were returned to quarantine, opened in isolation, fed fresh Tamarix foliage, and then were each separated into two approximately equal groups.One group from each shipment was maintained in the Albany quarantine for inspection, biological studies and colony development, and the other was shipped on to the ARS Quarantine Facility in Temple, TX. Additional incoming materials were also received from Temple, TX and the field nursery cages in Publo, CO. Voucher specimens were maintained from all batches of insects that were received and shipped.
 
All adult beetles were maintained in quarantine in standard sleeve cages stocked with Tamarix cuttings grown in the local greenhouses.Fresh host plant material was provided when needed as well as plant cuttings, which served as both a food source and an ovipositional substrate. Eggs were removed frequently (often daily), surfaced sterilized with a 10% Clorox Bleach solution for one minute, followed by three rinses in clean water. Several shipments of eggs were made to the Nevada nursery cages and for other field cage releases. Additional surface sterilized eggs were released from quarantine to the permitted ARS laboratory facilities in both Albany, CA and Temple, TX for additional evaluations.

At the Albany laboratory, additional host range studies were completed on several plants of agricultural importance to some of the biological control release areas. Grapes, sunflower, walnuts, lettuce, cucumber, plums, tomato, almonds and wheat were tested and found not to support adult feeding, oviposition, nor larval feeding (both small and large larvae tested). Additional research has focused on temperature dependent growth rates of larvae, rearing systems for Diorhabda larvae on individual Tamarix seedlings, and on other non-target feeding studies such as Tamarix aphylla and Frankenia salina.These studies are still underway and data were not yet available for summary.

The Temple, TX Quarantine Facility

(Submitted by Jack DeLoach and James Tracy, USDA-ARS, Temple)
 
The Temple laboratory received 3 shipments of D. elongata from Fukang, China, totaling 450 adults in May and 1,000 adults in July via the Albany Quarantine facility. Nursery cage and lab colonies begun from these adults at Temple were used to supplement populations at the Seymour, TX release site and to conduct life history studies in the laboratory. Survival, fecundity and life table data were obtained from D. elongata (Fukang, China) reared on four species of Tamarix naturalized in the U.S. (T. ramosissima, T. parviflora, T. canariensis, and T. aphylla), including 2 T. ramosissima accessions (additionally testing old and young foliage separately for one accession). Mean survival from egg to adult on potted plants differed significantly between treatments, ranging from 53% on T. canariensis to 91% on T. ramosissima, Lovelock, NV. Mean fecundity of adults fed excised leaves also differed significantly, ranging from 81.4 eggs on T. aphylla to 194.5 eggs on T. ramossima, Lovelock, NV (maximum 550 eggs was laid by one female reared on this accession). All Tamarix spp. were able to support comparatively similar, although significantly different, rates of population growth in the laboratory, with population doubling times ranging from 6.2 days on T. ramosissima from Lovelock, NV to 8.0 days on T. aphylla.Experiments at Temple to examine reproduction and dormancy are described in the Summer Aestivation and Over Wintering Diapause section (above).
 
Temple received two shipments of D. elongata from areas near the cities of Chilik and Chundzha, Kazakhstan, 1,900 live adults in early June and 138 live adults in early August. In June, 200 adults were sent to the Albany facility. Separate nursery cages were established at Temple for these beetles. No-choice host suitability studies were conducted in June by holding individual neonate larvae from Chilik in 10-dram plastic snap-cap vials with excised leaves of various plants. Results were similar to past tests of China (Fukang) populations.Survival to adult ranged from 33 to 83% on various Tamarix spp. and accessions, but no survival was noted past the first instar for any non-Tamarix spp. (including Frankenia spp.). In August, eggs, larvae and adults from nursery cages and lab colonies were shipped to the field cage at Delta, Utah. 

Fort Hunter Liggett, Monterery Co., California.

(Submitted by Ray Carruthers, USDA-ARS, and Tom Dudley, University of California, Berkeley)
 
Four hundred adults of Diorhabda elongata were introduced into a 12-ft. x 20-ft field cage at Fort Hunter Liggett on August 11, 1999.  The release site was adjacent to San Antonio Creek where the field cage was situated to contain mature saltcedar plants that were cut to fit within the cage.The beetles were dispersed throughout the cage by Art Hazebrook who is the Land Rehabilitation Coordinator for the Center for Ecological Management of Military Lands on Fort Hunter Liggett. Released beetles were observed moving onto the Tamarix foliage, and they began feeding immediately. Upon returning to the release site on August 21, significant defoliation was found in the southeastern corner of the cage, primarily on the tips of branches. However, only two living adults were found on the Tamarix plants within the cage on that date. Six other beetles were found stuck to insulation materials (alive but immobilized) and no other beetles were detected in the litter or on the soil surface. The cage was closely inspected, no signs of escape were detected, and no beetles were found on Tamarix plants outside of the cage. Upon further inspection on August 29, adult beetles were found inside the cage but down in the soil around the base of the plants (the soil is very sandy at this site). We believe that the Diorhabda elongata at this release site entered an aestivation or summer diapause period due to the 100°F+ temperatures that existed at this site during this period. Similar adult quiescence was also observed at the other California release site (Owens Valley), however, some larval development was detected in the Owens Valley site while none was observed at Fort Hunter Liggett. No beetles have been detected at Fort Hunter Liggett on the plants or in the soil from August 29 through the last monitoring period on November 26.

Independence, California

(Submitted by Ray Carruthers, USDA-ARS, and Tom Dudley, University of California, Berkeley)
 
Two 12 ft. x 20 ft. cages were erected near the City of Bishop, CA adjacent to the Owens River. Medium sized Tamarix plants were topped and caged to provide habitat and feeding sites for both sleeved eggs and larvae in one cage, and free ranging adults (plus additional eggs) in the other cage. In the sleeve cage release, 10 organdy sleeves each containing 20 eggs were secured on saltcedar branches on August 2, 1999. The insects within these cages were monitored approximately twice per week throughout the period from August 3 to the end of September 1999. Stage specific phenology patterns and estimated stage specific mortality should be calculable from these data once they have been corrected for differential temperature conditions. However, the estimated survival rate of these insects from egg to adult was approximately 20%. Survival rates on open foliage may either be higher or lower.It is currently unclear how much mortality may have been caused by high temperatures and humidities inside the organdy sleeves (especially when the foliage grew rapidly and filled the cages) or how much predation naturally occurred outside of the cages by spiders and ants. Some sleeves became infested with ants and all larvae were killed in these sleeves, however, outside of the sleeves, larvae were often seen dropping from the foliage when approached by ants. Adult beetles were occasionally seen successfully attacked by spiders (again, no quantification of this predation). In early October, all the insects that remained in sleeves were released into the large field cage for over-wintering.
 
In the open release field cage, 500 adult beetles and 200 eggs were released into the cage also on August 3, 1999. The insects were dispersed into the cage by Paula Hubbard of Los Angeles Water and Power, and by Bob Pierce of the Inyo Co. Agricultural Commissioner's Office. Again, the beetles dispersed throughout the Tamarix foliage and immediately began feeding. The adult beetles within this cage also followed the same pattern as seen at Fort Hunter Liggett. That is, after the first week, very few adult beetles could be seen on the foliage, presumably due to high temperature conditions. However, one or two adult beetles were found in the samples several weeks after the release. Search of the liter area around the Tamarix plants easily allowed detection of many beetles at the litter-soil interface. The soil at the Owen's Valley release site was much harder than at Fort Hunter Liggett and thus it seems that the beetles did not dig into the soil but remained in the litter. They were detected in these sites until the last monitoring period in October of 1999. No effort was made to quantify the number of beetles in the litter as we were afraid to disrupt their quiescence. Larval development from either one or the combination of the original eggs placed in the cage or from ovipositing adults produced a full larval generation within the free release cage. The peak larval occurrence was found 28 days following the original release with over 40 individuals detected on 12 sample branches. Further analysis will allow us to estimate the number of larvae in the entire cage and to determine if some oviposition did occur at the Owens Valley release site.

Pueblo, Colorado

(Submitted by Debra Eberts, USDI Bureau of Reclamation, Denver)
 
The first release of Diorhabda eggs into the secure nursery cages occurred 6/24/97. While these eggs produced adults, no second generation resulted. The low numbers of insects (less than 30 adults) was probably the reason no insects survived the winter. Another attempt was made, with more eggs shipped on May 29, 1998 and June 2, 1998. The 181 adults produced a second generation, and enabled over-wintering to be successful. These over-wintered adults (about 130 survivors) emerged from hibernation under the litter and straw in the tents at the beginning of May 1999. Feeding by the adults and larvae destroyed more than 90% of the foliage by mid-July, and over 3,000 first generation adults were removed and transferred to other secure field tents and to researchers in other states. Egg laying was evident throughout July and August, but mostly ceased after the beginning of September. A second generation of adults emerged in the beginning of September and is expected to over-winter successfully.
 
Foliage damage caused by Diorhabda was significant, but it was matched by the foliage damage caused by leafhoppers (Opsius stactogalus). The leafhoppers have little effect on trees outside the secure field tents, but inside the cages, they cause significant defoliation of the trees. This has caused complications in researching the effects of Diorhabda, as the results are confounded, and the reproduction of Diorhabda is limited by lack of food. These leafhoppers are competitors, not predators, of Diorhabda. However, at this site they caused greater mortality than the jumping spiders or ants.

Lovelock and Schurz, Nevada

(Submitted by Jeff Knight, Nevada Division of Agriculture, Reno) 
 
During 1999, no over-wintering adults (from 1998) were observed in the nursery cage at Lovelock. During July, 1999, approximately 500 eggs were placed in bags in the original cage at Lovelock. This cage was visited weekly and observations taken on hatching development and larval numbers. Adults were observed in this cage. However, accurate counts could not be made due to an excess of foliage in the cage (one of the problems this year even though the plants were pruned during the winter). Eggs and larvae were observed from these adults however.Although no environmental recordings were taken during this time, temperature data is available from a nearby airport. A shipment of 300 adults were received from the Pueblo, CO site and placed in bags in the second cage at this site in mid-July. These appeared to lay eggs and behaved “normally.” Eventually adults were produced from these eggs. Once the permit was approved, some of this shipment was transferred to a cage at Schurz, near the Walker River on the Native American Walker River Paiute Reservation.These adults also laid eggs. Adults from these eggs were observed at the Schurz site. A second shipment of 200 adults was placed in bags at Lovelock then transferred to Schurz also. These however laid very few eggs at either site and appeared to go into aestivation.
Predators continue to be a problem. Jumping spiders (Salticidae) were probably the most common and one was observed eating a full-grown larva. Predators were killed whenever possible. Very few predators were observed in the original cage in the second year. However, leafhoppers did defoliate this cage very late in the summer. (A side note on leafhoppers... we appear to have had very high populations of these around western Nevada the last couple of years and they possibly even caused some Tamarix die back at Schurz. In the future, new cage sites, if needed,will be fumigated prior to use).

Seymour, Texas

(Submitted by Allen Knutson, Texas Agricultural Experiment Station, Dallas, and Phil Lewis, USDA-ARS, Temple, TX)
 
The first release of Diorhabda eggs and larvae into a secure field cage occurred 7/21/99.Adults were released within cages on 7/27/99 and 8/2/99. Predation of the adult beetles by spiders and assassin bugs was evident and leafhoppers (Opsius spp.) reduced foliage quality during the first month of the release only. Released larvae and those coming from eggs were seen to mature to the third instar within the cage and likely pupated successfully, although no new generation adults were observed on the foliage. No evidence of adult egg laying was ever observed and released adults became inactive and absent from the foliage. Upon searching, several adults were found aestivating on the soil beneath a mat of dead grass. Monitoring will resume in the spring when we expect these over-wintering adults to emerge and become active.
 
A second cage was prepared at this site and was treated with a pesticide fog for leafhopper control in late July.One month after this, on August 27, a small number of 1st instar larvae was released in this cage. Larvae were observed to develop to later stages and pupation occurred in mid-September but no adults were observed in this cage.

Temple, Texas

(Submitted by Jack DeLoach, Phil Lewis, James Tracy, Tom Robbins, and Joye Johnson)
 
Temple, TX is an approved nursery cage site, not requested as a field release site, since no saltcedar grows nearby. On 18 June, 1997, the first D. elongata nursery cage was started at Temple, TX. From the less than 70 first generation eggs of a Chilik, Kazakhstan population reared in sleeve cages, only 13 first generation adults (9 of these deformed and weak) were produced which emerged ca. 22 July. These and 14 other quarantine-reared adults placed in the cage from 24-29 July were never observed to lay 2nd generation eggs and they disappeared by August 5, when high leafhopper populations were also noted. No beetles were observed to over-winter.
 
In 1998, nursery cages were established for D. elongata from Fukang, China and Chilik, Kazakhstan. In the China nursery cage, about 350 eggs were introduced on May 29 and ca. 10 first generation adults began appearing June 22. Because 12 lab-reared second-generation adults were added on 22 July, it was unclear when, or if, second generation adults were produced in the field. The last eggs were laid in the cage on July 30. Eggs from a second laboratory generation placed in the cage in early August appear to have produced a third generation of about 25 adults that emerged in early September. These adults did not reproduce and disappeared by September 20. Leafhopper damage was evident in late July and plants were totally brown by September 14, but most plants had begun re-foliating by October 14. In the Kazakhstan nursery cage, about 50 first generation adults were produced around August 1 from 206 eggs and 50 third instar larvae placed in the cage from July 6 -15. These adults laid no eggs and disappeared by 17 August. Leafhopper damage was not significant in this cage. No adults were observed to emerge from over-wintering the next spring from either the China or Kazakhstan cage.

In 1999, five field cages received varying numbers and stages of D. elongata, from Chundzha and Chilik, Kazakhstan (2 cages) and Fukang, China (3 cages), from late May to September. One cage with beetles from China and one cage with beetles from Kazakhstan produced second generation. These two cages had 700 to 1,000 eggs and first instar larvae introduced per cage from late May to early June but produced only 90 to 180 second generation adults that emerged in early to mid August. The small size of some Tamarix plants and heavy leafhopper populations in the cages probably limited the amount of young foliage that appears to be most suitable for the beetles. This also may have contributed to an estimated > 50% mortality between third instars and adults. In two cages with low leafhopper populations in June and July, third instar Diorhabda larvae completely defoliated some branches. However, the Diorhabda damaged plants re-foliated during August and September when beetles almost completely ceased to lay eggs. In one China cage, first generation adults began appearing on June 24; peak egg laying occurred on July 8, when approximately 50 adults were seen on the foliage. Maximum temperatures during peak egg laying ranged from ca. 33 to 35.5oC during the several days around July 8. In this cage, peak emergence of second-generation adults was observed on August 3 (89 adults), a week when maximum temperatures ranged from about 36.5 to 37.5oC. These beetles disappeared without reproducing over the next two weeks, when the maximum temperature was 39.2oC. Beetles emerging after early August in all cages usually disappeared within about 2 weeks without reproducing, apparently being reproductively dormant. No adults were produced from over 1,000 first instar larvae placed in a field cage in early September, perhaps due to poor quality foliage and low quantities of young foliage. On November 12, a small number of over-wintering adults were found in the grass/leaf litter of one China cage, but searches failed to locate beetles in the other four cages.D. elongata did not feed or oviposit upon any plants other than saltcedar in the nursery cages, including cages with large plants of Goodding willow (Salix gooddingii) and Coyote willow (S. exigua). Additional studies are discussed in the main report above.

Delta, Utah

(Submitted by Greg Abbott, USDA-APHIS, Richfield, and Pat Fosse, USDI-BLM, Filmore)
 
The first release of Diorhabda into a field cage along the Sevier River occurred on 8/5/99. This consisted of 200 second and third instars larvae originating from Chilik, Kazakhstan, which were released into cage #1. On 8/20/99, 100 adult beetles, also originating from Kazakhstan, were released into cage #1. Though the leaf litter in the cage was never disturbed to look for pupae, it appears that the original larvae did pupate and emerge as adults. No eggs or additional larvae from the two introductions were observed before the advent of colder weather.
 
The first release of Diorhabda eggs, originating from Kazakhstan, occurred on 8/20/99. We placed 107 eggs into 8 sleeves in cage #2 and monitored these through pre-pupation. At that point the larvae were released and allowed to pupate in the leaf litter of the cage. On two occasions, young instar larvae were found to have escaped from the sleeves, which may have artificially lowered survivability counts (27%) therein.  Foliage damage from Diorhabda inside the cages and the sleeves was insignificant probably due to the relatively low population density of beetles and from lack of reproduction this year. Leafhoppers (Opsius stactogalus) never achieved numbers that significantly affected saltcedar foliage within the cages, though population increases became dramatic by the end of August.

Lovell, Wyoming

(Submitted by Dave Kazmer, University of Wyoming and Gary Adams, USDA-APHIS, Billings, MT)
 
Two hundred adult Diorhabda elongata were released into each of two secure field cages near the Bighorn River on July 22, 1999. Eggs and larvae of D. elongata were subsequently observed in both cages but the numbers of larvae were significantly greater in Cage B than in Cage A. Ultimately, only Cage B produced a new generation of adults. New generation adults were first observed approximately six weeks after release. We suspect that the different numbers of larvae and adults between the two cages is due to different levels of predation on D. elongata by jumping spiders, ants, and possibly other generalist predators.
 
Leafhoppers became very abundant in Cage B (but not Cage A) near the time of emergence of new generation adults. Because of the extensive amount of foliar damage caused by the leafhoppers in Cage B, a third field cage was erected and 100 adults were transferred to that cage. Little feeding and no egg laying by the new generation adults were observed. Between Cage B and the new cage, at least 100 adults entered the over-wintering period.

Last Modified: 6/4/2008
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