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A physiological age-grading system for female Hydrellia pakistanae Deonier (Diptera: Ephydridae)
Biological Control 42 (2007) 119–128 www.elsevier.com/locate/ybcon
A physiological age-grading system for female Hydrellia pakistanae Deonier (Diptera: Ephydridae) Jennifer M. Lenz b
a,1
, Michael J. Grodowitz
b,*
, James H. Kennedy
a,1
a University of North Texas, Denton, TX 76203, USA US Army Engineer Research and Development Center, Vicksburg, MS 39180, USA
Received 5 September 2006; accepted 23 March 2007 Available online 24 April 2007
Abstract The female reproductive system of Hydrellia pakistanae Deonier was described by dissecting individuals of known ages and describing changes in relation to number of eggs oviposited. Female H. pakistanae have meroistic ovaries, in that specialized nurse cells or trophocytes (nutritive cells associated with the eggs) are present within each ovariole, and of the polytrophic subtype because nurse cells closely accompany each developing egg, or oocyte. The reproductive system includes two ovaries, each consisting of 8 or 10 tube-like ovarioles. The ovariole can be divided into two distinct areas: a distal germarium, which produces the follicles, and a more proximal vitellarium, which houses the developing follicles. Each ovariole is surrounded by an ovariole sheath and houses several developing follicles, a term referring to the ova (yolk and surrounding cells; i.e., the egg), nurse cells, and surrounding epithelium. Within an ovariole, the follicle proximate to the lateral oviduct is the most mature with each subsequent, more distal follicle becoming less mature. All ovarioles within an ovary are connected via the lateral oviducts, while the common oviduct connects the ovaries to each other. The spermatheca and accessory glands branch off the common oviduct. Sperm stored in the spermatheca fertilize eggs as they pass through the common oviduct. Based on this information the continuum of ovarian maturation was divided into three nulliparous and four parous stages. The nulliparous stages are classified based on degree of ovariole segmentation and maturity of the most proximal follicle while follicle relic quantity and appearance is mainly used to separate the parous classes. Limited field sampling demonstrated the possibility of using this system to assess reproductive health of wild populations. 2007 Elsevier Inc. All rights reserved. Keywords: Hydrellia pakistanae; Hydrilla verticillata; Biological control; Age-grading; Follicular relics
1. Introduction Hydrellia pakistanae Deonier, an introduced leaf-mining fly species in the family Ephydridae, is a potentially valuable organism for the biological control of Hydrilla verticillata (L.f.) Royle, an invasive aquatic plant. Native to Pakistan, India, and as far north as Beijing, China, H. pakistanae was first released in southern Florida in 1987. Currently, it has been released at over 50 sites in Alabama, *
Corresponding author. Fax: +1 601 634 2398. E-mail addresses: [email protected] (J.M. Lenz), [email protected] (M.J. Grodowitz), [email protected] (J.H. Kennedy). 1 Fax: +1 940 565 4297. 1049-9644/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2007.03.015
Arkansas, California, Florida, Georgia, Louisiana, North Carolina, Virginia, and Texas (Center et al., 1997). H. pakistanae is now established in Arkansas, Louisiana, Florida, Georgia, and Texas (Center et al., 1997) with range expansions noted in both Texas and Louisiana (Grodowitz et al., 2000; Julie Nachtrieb, US Army Engineer Lewisville Aquatic Ecosystem Research Facility, Lewisville, TX, personal communication and Jan Freedman, US Army Engineer Research and Development Center, Vicksburg, MS, personal communication. The larvae of H. pakistanae feed exclusively on hydrilla, and while they have been shown to impact photosynthesis and cause significant damage to experimental populations (Doyle et al., 2002, 2007), there are conflicting opinions as to their impact on field populations of hydrilla. H. pakistanae appears to be successful at
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controlling hydrilla in Texas field populations (Grodowitz et al., 1999); however, fly populations at many Florida sites have only achieved a maximum of 15% leaf damage to hydrilla, with many sites exhibiting much lower population levels (<200 immatures/kg) and associated damage (Wheeler and Center, 2001). Recent work by Grodowitz et al. (2004) has characterized the impact of the fly to both experimental and field populations. Reasons for such low population levels at many sites are unknown but several ideas have been cited. These include parasitism by a native pupal parasite (Trichopria columbiana Ashmead (Hymenoptera: Ichneumonidae) (Wheeler and Center, 2001; Doyle et al., 2002; Christie Snell and Robin Bare, US Army Engineer Lewisville Aquatic Ecosystem Research Facility, Lewisville, TX), plant nutritional changes influencing larval development (Wheeler and Center, 1996; Michael Grodowitz, Jan, Freedman, and Dwilette McFarland, unpublished), and high temperatures. While some of these ideas have been studied to an extent, one possible mechanism for low population development has been poorly addressed and only rarely mentioned as a possible mechanism. This is the impact adult nutrition has on reproductive development, egg production, and oviposition rates. Only minimal information is available on the reproductive development of H. pakistanae, let alone adult nutritional requirements. Most of the present literature focuses on its release, establishment, and success as a biological control agent. The first step toward adequately studying adult H. pakistanae nutritional requirements is to provide information describing, in detail, basic reproductive morphology in females as well as the overall development of the reproductive system through time. This is an important prerequisite to understanding the influence adult diet has on egg production and oviposition rates. Published accounts are of little help; only one paper describing the female reproductive system exists (Deonier, 1971) and only minimal morphological details are included in this paper. To address this gap in the literature, the research described here provides details of morphology of the female reproductive system and associated changes through time for H. pakistanae. It describes a physiological age-grading system based on changes in the female reproductive system. Such a system will be important for understanding and measuring changes in reproduction in relationship to adult nutritional requirements. 2. Materials and methods 2.1. Source of insects Rearing methods are modified only slightly from those found in the literature (Freedman et al., 2001). H. pakistanae larvae and adults were collected from colonies maintained at the Lewisville Aquatic Ecosystem Research Facility (LAERF) in Lewisville, Texas, and from colonies established in the Department of Biological Science at the
University of North Texas (UNT) in Denton, Texas. Both colonies consisted of immature rearing containers and oviposition boxes. Rearing containers were 4-l in volume and filled with about 75% hydrilla in water, leaving enough headspace for adults to mate and oviposit. Adults were periodically removed from the containers using an aspirator and sorted by sex and species. Those adults not used for experiments or dissections were placed into the oviposition box to encourage additional ovipositions. Oviposition boxes were slanted with a height measurement of 54.5 cm (back), 47 cm (front), and a depth of 63.5 cm. The front, measuring 122 cm across, had two circular openings allowing for internal access, each fitted with a cloth sleeve to prevent flies from escaping. The top of the oviposition box was constructed of Plexiglas to allow maximum light penetration. In each oviposition box, small petri dishes with several drops of two adult food mixtures and large petri dishes with hydrilla stems in water for use as an oviposition substrate were included. Immature larvae found on the hydrilla were placed back into the rearing containers to enlarge the colonies. Adult food consisted of a sugar–yeast hydrolyzate mixture (4 g yeast hydrolyzate, 7 g sucrose, 10 ml water) and a sugar–water mixture (1:1) (Freedman et al., 2001). Food was changed frequently to prevent formation of fungal contamination. Adults for field studies were collected from the surface of experimental ponds containing hydrilla at the LAERF using a hand-held, battery-powered insect vacuum. Flies were collected during the active growing period, i.e., June through September. 2.2. Dissection techniques Females were dissected alive using a stereomicroscope (25–35·) to prevent deterioration of internal organs that occurs shortly after death. Adults were held individually in plastic vials at room temperature until just prior to dissection. Adults were anesthetized by placing them briefly in a freezer (3–5 min) or by exposure to CO2. Inactive flies were removed and immediately pinned through the dorsal surface of the thorax; ventral side down, onto an electron microscopy stub covered with a mixture of beeswax, paraffin, and dark-colored crayon wax. The flies were covered completely with a phosphate buffered saline, pH 7 (PBS) in an effort to protect the ovaries from desiccation and deterioration due to osmotic changes. Grasping the abdominal cuticle along the mid-dorsal line with fine forceps and gently tearing the cuticle opened the abdominal cavity, exposing the ovaries. Prior to removal of the ovaries, characteristics of the fat body (if visible) were noted. Ovaries were carefully removed by grasping the common oviduct using fine forceps. The ovaries were subsequently placed onto a microscope slide in a drop of PBS for examination. The ovaries were first viewed using a dissecting microscope, and examined under a compound microscope at 100· and 200· magnifications to distinguish changes in reproductive system morphological markers. Characteris-
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tics such as the quantity and quality of follicular relics, number of ovarioles per ovary, number of mature ovarioles, and the condition of the ovaries and spermatheca were noted. Photomicrographs were taken at different magnifications under both dissecting and compound microscopes for later comparisons. 2.3. Baseline studies Hydrellia pakistanae females randomly collected from the rearing containers were used to practice dissection techniques and describe reproductive morphology. Chronological age, if known, was used to associate general reproductive changes over time. In order to associate physiological changes in the reproductive system with fecundity (number of eggs oviposited), females of a known age that had not yet oviposited were used. Two techniques for collecting young females were used. In one technique, young females (±0.50- to 0.75-h-old) were obtained from colonies by first removing all adults from the rearing containers. After 1 to 1.5 h, all newly emerged adults were collected. After this time, the females collected were not used, unless males were completely absent. While females often emerge with an almost fully mature reproductive system, no eggs are oviposited until mating is completed (personal observation). A second technique was developed to collect young females from field populations. Hydrilla stems with obvious fly damage were collected from experimental ponds at the LAERF. Pupae were removed (while still attached to the stem) and placed individually into vials with enough water to cover the stem. Since each pupa was isolated, it was not necessary to ensure the females were within 1 to 1.5 h old because no mating, and hence oviposition, could have occurred. H. pakistanae females collected via either technique were held in a plastic vial while a 240-ml holding container was readied. Each container held several short hydrilla stems in 30 to 60 ml water for oviposition, and a 1-cm2 floating piece of Styrofoam carrying one drop of each of the two adult food mixtures. The females were placed individually into the containers, and one or two males were added as soon as they were available. The containers were maintained in an incubator at 24–25 C with a photoperiod of 18 light: 6 dark and a relative humidity of about 50%. The containers were checked daily, and hydrilla stems removed and carefully inspected for eggs. Eggs were tallied and whorls containing eggs were removed to prevent re-counting. Each stem was checked twice if the female was about to be dissected. For females that were being held for a longer period, hydrilla stems without eggs were placed back into the cup so that any missed eggs could be enumerated at a later time. Fresh food was added and adults replaced. When larger numbers of eggs were needed, females were not handled for the first two days. This seemed to encourage additional ovipositions. Males were replaced if found dead because females apparently required their presence to continue ovipositing. If, upon
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dissection, an egg was found in the lateral or common oviduct, it was also counted since the epithelium and nurse cells that form relics had already been left behind. Dead females were dissected only if evidence indicated a recent death (within the hour). In this case, the data were only used if the ovaries showed no signs of deterioration and then only to corroborate other data in the same class. Researchers have shown (Buckingham and Okrah, 1993; Buckingham et al., 1989) that females feeding on the diet used in this study oviposited an average of 68 eggs. Hence, an attempt was made to observe females that had oviposited at least this many eggs. Females were observed that oviposited a total of 68 eggs (i.e., 3 or 4 full-batches depending on the number of ovarioles per ovary). It was difficult to get higher egg counts because it appeared that isolation and frequent handling caused females to lay fewer eggs. Although female H. pakistanae have been shown to oviposit >100 eggs under laboratory conditions (Buckingham and Okrah, 1993), it is believed that the appearance of the follicular relics at higher egg counts would not differ greatly from that seen in the highest class observed in this research. Space in the lateral oviduct for the relics to accumulate is limited, so after relics reach the size of clusters found in the highest class, they will most likely only become denser and darker. Approximately 200 H. pakistanae females were dissected for the baseline studies. Most of the flies were used to become familiar with the overall reproductive morphology and morphological changes that occur through time. More than 50 females were kept under the controlled conditions described earlier. The number of eggs oviposited and reproductive characteristics upon dissection were recorded for these females. These data were used to develop the physiological age-grading system by associating changes in the reproductive system with the number of eggs ovulated and oviposited. 2.4. Field studies The developed physiological age-grading system was tested on field populations at the LAERF experimental ponds. Flies were vacuumed from the surface of several ponds on five different days during June 2002. Collected Hydrellia spp. were identified to species by examining external genitalia. H. pakistanae females, distinguished by their L-shaped cerci, were dissected using the methods described above. The flies were placed into classes according to the developed physiological age-grading system. A total of 56 females were dissected. 3. Results and discussion 3.1. Reproductive morphology The H. pakistanae female reproductive system is as described by Deonier (1971) for Nearctic Hydrellia. It is meroistic, in that specialized nurse cells or trophocytes
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(nutritive cells associated with the eggs) are present within each ovariole, and of the polytrophic subtype because nurse cells closely accompany each developing egg or oocyte (Chapman, 1982). The reproductive system includes two ovaries, each consisting of 8 or 10 tube-like ovarioles (Fig. 1). The ovariole can be divided into two distinct areas: a distal germarium, which produces the follicles, and a more proximal vitellarium, which houses the developing follicles (Fig. 2a). Each ovariole is surrounded by an ovariole sheath and houses several developing follicles, a term referring to the ova (yolk and surrounding cells), nurse cells, and surrounding epithelium (Fig. 2b). Within an ovariole, the follicle proximate to the lateral oviduct is the most mature with each subsequent, more distal follicle becoming less mature. A mature follicle (one that is ready to be ovulated) appears full with yolk and has no visible nurse cells. An immature follicle has visible nurse cells with variations in yolk amount (Fig. 2b). All ovarioles within an ovary are connected via the lateral oviducts, while the common oviduct connects the ovaries to each other. The spermatheca and accessory glands branch off the common oviduct (Fig. 1). Sperm stored in the spermatheca fertilize eggs as they pass through the common oviduct. Maturation of proximate follicles (those closest to the lateral oviduct) appears to be independent between ovarioles. In other words, once a follicle is ovulated (i.e., passes to the lateral oviduct), the next distal follicle will begin to mature regardless of the maturity of the proximate follicles
in other ovarioles within the same ovary. Evidence of this is seen in ovaries that have proximate follicles of various states of maturity. For example, one ovary may have a single mature follicle as well as follicles of varying maturities, all of which are immediately distal to the lateral oviduct (Fig. 2c). Another example is when all the proximate follicles are immature, but one stands out as distinctly closer to maturity than the others (Fig. 2d). 3.2. Fat body The fat body is also used to determine physiological age. It is easily identified in the abdominal cavity and in recently emerged females the fat body appears diffuse and green (Fig. 3a and b) and becomes clustered and green within 1–2 h after emergence (Fig. 3c and d). The green color is apparently derived from pigments within hydrilla tissue consumed during the larval stages. Eventually the fat body changes color becoming milky white and minimal in quantity, making it difficult to observe. Changes in the fat body are only useful in distinguishing nulliparous classes (those where no eggs have been oviposited) and those classes with low egg counts because it does not change distinctly after oviposition begins. 3.3. Follicular relic formation As an oocyte passes through the lateral oviduct (i.e., ovulation), the surrounding epithelium and nurse cells slough off and accumulate within the base of the lateral
Fig. 1. Photomicrograph of the reproductive system of H. pakistanae showing the overall structure of each ovary, connecting oviducts, and associated glands. This example is from a female with eight ovarioles per ovary.
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Fig. 2. A composite plate with images depicting various morphological features of the H. pakistanae ovaries (a and b) as well as differences in follicle maturation (c and d). (a) Close-up of a single ovary from a female with 10 ovarioles per ovary showing the two distinct regions associated with follicles; i.e., the germarium (g) and vitellarium (v); (b) morphological features of a single immature follicle; (c) ovary having proximate follicles in various states of maturity—in this case there are even number of immature and mature proximate follicles as compared with plate (d), where most follicles are immature but one is distinctly more mature (d).
oviduct. These accumulations of cellular debris are known as follicular relics. As more and more cellular material is sloughed off in the lateral oviduct, the follicular relics become compressed and hence change in appearance, allowing for an estimation of fecundity. Two distinct types of relics are observed: brown follicular relics that are relatively loose in the ovariole sheath (Fig. 4a), and yellow follicular relics, which appear compressed and granular or particulate in the lateral oviducts (Fig. 4b). The brown follicular relics are presumably epithelium and nurse cells recently left behind after an ovulation, while yellow follicular relics are composed of the same
cellular remnants, only compressed, indicating that subsequent ovulations have taken place. Both types of relics may be found simultaneously. Characteristics of the yellow follicular relics can be used to estimate physiological age. Brown relics indicate a recent ovulation and can obscure the presence and appearance of the yellow relics. Yellow follicular relics are diffuse and scarce in females that have oviposited few eggs, but gradually become larger and more clustered with successive ovulations. The color of the yellow relics also appears darker with more ovulations due to the accumulation of material and compression of the relics. After an ovulation, the yellow relics can appear
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Fig. 3. Photomicrograph of diffuse green fat body seen in the abdominal cavity (a and b) contrasted with clustered fat body in (c and d).
Fig. 4. Photomicrograph of brown follicular relics (B) loose in the ovariole sheath with compressed yellow relics (Y) below (a). In (b), yellow follicular relics can be seen compressed in the lateral oviduct of an ovary.
particulate because they become broken apart by the passing oocyte. The volume in the lateral oviduct composed of yellow relics generally increases with a higher number of ovulations, but apparently is not reliable in determining fecundity. This occurs because some of the preexisting yellow relics are apparently flushed out of the lateral oviducts along with the egg during successive ovulations. Size of relics may also be reduced due to compression by passing fol-
licles. As evidence of this, no significant correlation (n = 45, P > 0.05, r = 0.259) between relic size and number of ovipositions was noted. 3.4. Physiological age descriptions Physiological age classes were based on changes in the morphology of the female reproductive system and allow for a rough estimation of the number of eggs oviposited
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(Fig. 5). Classes were identified using characteristics of both the fat body and follicular relics (Table 1). Two broad class types were noted: nulliparous classes (i.e., N1–N3), where no eggs have been oviposited, and parous classes (i.e., P1–P4), where eggs have been oviposited. 3.4.1. Nulliparous classes 1–3 (N1–N3) Females in these classes have deposited no eggs and the ovaries are distinguished by the absence of follicular relics. In N1, the fat body is highly diffuse and green, and takes up more than half of the abdominal cavity. None of the proximate follicles are mature (Fig. 6a). In Grodowitz et al. (1999) and Van Geem et al. (1983), the ovarioles in the N1 stage were described as being totally nondifferentiated; i.e., being without distinct follicles. This was never observed in H. pakistanae presumably because nondifferentiated ovarioles occur before adult eclosion and typically will not be observed under field conditions. In the N2 class, the fat body also occupies more than half of the abdominal cavity, but appears as distinct green clusters. Some of the proximate follicles are mature (Fig. 6b). N3 is the class just Fig. 6. (a) Photomicrograph of an ovary with no follicular relics and no mature follicles representing nulliparous class 1 or N1. (b) Photomicrograph of a pair of ovaries with no follicular relics and some mature follicles; i.e., three mature follicles in the right ovary and two in the left. This female represents nulliparous class 2 or N2 (b).
before ovulation occurs. In this class, the fat body is very similar to that of N2, but now most of the proximate follicles are fully mature and ready to be ovulated but no follicular relics are present.
Fig. 5. Mean number (±1 standard error and 95% confidence interval) of eggs oviposited by females in each parous class (P1–P4).
3.4.2. Parous class 1 (P1) Individuals in the P1 class have oviposited from one to about 15 eggs (Fig. 5). The fat body may still be present as distinct green clusters, but occupies less than half the abdominal cavity, or has become milky white and scarce. Proximate follicles may all be mature, all immature, or with some variable number of each. This type of variation is
Table 1 Physiological age classes and associated characteristics for H. pakistanae females Class
# eggs
Fat body
Ovariole condition
Follicular relics
N1 N2 N3
0 0 0
Dense, green Clustered, green Clustered, green
None mature Some mature All mature
None None None
P1
1–15
Scarce (color varies) or absent
None, some or all mature
Diffuse both ovaries, with or without brown relics Mostly diffuse with small clusters in one ovary, diffuse in other ovary
P2
15–40
Scarce (color varies) or absent
None, some or all mature
Diffuse with small clusters both ovaries Light particles both ovaries
P3
40–60
Scarce (color varies) or absent
None, some or all mature
Partly diffuse or particulate with large particulate clusters, one or both ovaries Large particulate clusters both ovaries
P4
60+
Scarce (color varies) or absent
None, some or all mature
Large dense clusters both ovaries
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seen in all parous classes. It is possible to observe the presence of only brown relics (indicating one or two ovulations) but care must be taken not to miss yellow relics, which can be easily obscured by the brown relics. Relics in both ovaries are scarce, very light, and diffuse, with no brown relics present in the earlier stages of this class (Fig. 7a), or the same accompanied by brown relics towards the middle of
the class (Fig. 7b). In late P1 individuals, yellow relics are still mostly diffuse, but start to appear clustered in one ovary indicating subsequent ovipositions (Fig. 7c). 3.4.3. Parous class 2 (P2) Oviposition number ranges from the mid-teens to <40 for individuals in the P2 class (Fig. 5). The fat body in this
Fig. 7. Series of photomicrographs depicting the various parous classes. The first series of images (a, b, and c) represents parous class 1 (P1) as indicated by minimal quantities of diffuse follicular relics. The number of eggs oviposited by these three different females ranges from 3 to 14. Photomicrographs (d and e) show mostly diffuse yellow relics with only small clusters in both ovaries. These females oviposited from 16 to 35 eggs and belong in parous class 2 (P2). Photomicrographs in (f, g, and h) all show diffuse yellow relics with large particulate clusters in both ovaries. These females oviposited from 44 to 56 eggs and belong in parous class 3 (P3). Photomicrograph of a pair of ovaries showing large dense yellow relics in both (i). This female laid 62 eggs and belongs in parous class 4 or P4.
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class (and subsequent classes) is usually very scarce and may easily be missed. Yellow relics are still mostly diffuse, but begin to appear clustered in both ovaries and may or may not be accompanied by brown relics (Fig. 7d and e). It is possible for relics in one ovary to be light and diffuse, with the other ovary having relics that show signs of recent ovipositions, i.e., brown relics and particulate yellow relics. This is only seen in the earlier stages of this class. Both ovaries may also have yellow relics that appear particulate. 3.4.4. Parous class 3 (P3) Individuals in the P3 class have an oviposition range >39 and <60 (Fig. 5). In this class, the yellow relics appear as large particulate clusters, but are usually partially diffuse or particulate (Fig. 7f, g, and h). Occasionally, both ovaries have particulate yellow relics, where the particles are medium in color and take up a large amount of space in the lateral oviduct. This amount of space is about equal to the size of the yolk in a half-mature follicle (where the yolk equals half the total size of the follicle). 3.4.5. Parous class 4 (P4) This class contains individuals with ovipositions P60. The yellow relics begin to appear as large dense clusters. The dense clusters resemble the clusters in P2, but are larger and darker (Fig. 7i). Size of the clusters is comparable to the size of the circular nurse cells seen in immature follicles or it may be larger. This physiological age-grading system provides a rough approximation of ovipositions and is designed to distinguish females that have not oviposited or have laid only a few eggs from those that have oviposited higher numbers (Fig. 5). There is some degree of overlap of the characteristics found in each class and as ovipositions increase, variation of the characteristics associated with each class increases. For this reason, this system will provide only an estimate, but not an exact number, of oviposition numbers for field populations. 3.5. Field studies Females examined from experimental field populations at LAERF demonstrate application of the developed system. H. pakistanae females could easily be assigned to the described classes and the observed characteristics did not differ from those in the controlled baseline studies. No females were nulliparous, but this is not surprising because females will mate and oviposit within 1–1.5 h after emerging. Since the nulliparous classes apparently last for only a short time, individuals in these classes may be difficult to observe in field populations. Also, females that have recently emerged may not be as active and thus, not as likely to be collected. The lack of individuals in the nulliparous classes can be expected because it is typical to have under-representation in the youngest age classes (Hayes and Wall, 1999). Approximately 15% of the dissected field flies were classified as P1 (Fig. 8). Larger proportions (50%)
Fig. 8. Percent of total number of female H. pakistanae from LAERF experimental field populations in each parous class. The column between P3 and P4 represents those with characteristics of both classes.
of the flies were classified as P2. Since this class is rather large, and encompasses the middle of the physiological age distribution, this was not unexpected. Approximately 20% were classified as P3, while another 9% showed characteristics of both P3 and P4. These females most likely had oviposition numbers in the high 50 s or low 60 s. The remaining 7% of the females were classified as P4. This means that 7% of H. pakistanae females from the population sampled each laid 60 or more eggs; i.e., 3 or 4 full-complements (depending on the number of ovarioles per ovary). 3.6. Importance of physiological age-grading systems Physiological age-grading systems are useful, particularly for insects, where physiological (rather than chronological) age determines many characteristics, such as fecundity, certain behaviors, and lifespan (Tyndale-Biscoe, 1984). In the area of biological control, physiological agegrading systems allow researchers to estimate the future impact of a release, as well as to ascertain the success of previous releases. For example, successful biological control agents must be able to reproduce and buildup large populations to control the target pest. This can be assessed fairly easily using such a developed system. Physiological age-grading systems can also be used to track reproductive status of the biological control agent through time. This can provide helpful information such as peak reproductive activity of the released individuals. Determining physiological, rather than chronological, age is valuable because the two may differ considerably. While chronological age is solely based on time and is not influenced by outside factors, physiological age is based on number of eggs oviposited and is influenced by environmental factors such as climate and nutritional supply. Creating a physiological age-grading system for H. pakistanae using ovarian morphology to evaluate reproductive
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status of the fly in field populations will be useful in assessing impacts of H. pakistanae on hydrilla and measure the potential success of releases based on reproductive activity of the introduced individuals. Knowing the relative numbers of eggs being oviposited by females provides researchers an idea of the physiological age distribution of the fly. This information is useful in estimating size of future generations, and therefore possible impact on hydrilla. Although, the exact number of ovipositions cannot be determined, researchers can easily distinguish flies that have oviposited relatively few eggs from those that have oviposited many. This information will provide some evidence as to whether or not H. pakistanae are depositing enough eggs to maintain populations sufficient to control hydrilla. It will also give important clues to the varying success of H. pakistanae as a biological control agent for hydrilla. Acknowledgments This research was accomplished as part of the requirement for completion of a Masters Thesis of the senior author at the University of North Texas in Denton, Texas. The US Army Engineer Research and Development Center provided funding for this project under the Aquatic Plant Control Research Program, Dr. John Barko, Program Manager. The Lewisville Aquatic Ecosystem Research Facility in Lewisville, Texas provided use of experimental ponds, fly colony, and equipment. Thanks to Mses. Robin Bare, Christi Snell, Chetta Owens, and Jessica Sharp for technical assistance. Thanks are also extended to the author’s Thesis committee members, Drs. Ken Dickson and Tom Waller. References Buckingham, G.R., Okrah, E.A., 1993. Biological and Host Range Studies with Two Species of Hydrellia (Diptera: Ephydridae) that Feed on Hydrilla. Technical Report A-93-7, US Army Engineer Waterways Experiment Station, Vicksburg, MS. Buckingham, G.R., Okrah, E.A., Thomas, M.C., 1989. Laboratory host range tests with Hydrellia pakistanae (Diptera: Ephydridae), an agent for biological control of Hydrilla verticillata (Hydrocharitaceae). Environ. Entomol. 16, 164–171. Center, T.D., Grodowitz, M.J., Cofrancesco, A.F., Jubinsky, G., Snoddy, E., Freedman, J.E., 1997. Establishment of Hydrellia pakistanae (Diptera: Ephydridae) for the biological control of the submersed
aquatic plant Hydrilla verticillata (Hydrocharitaceae) in the southeastern United States. Biol. Control 8, 65–73. Chapman, R.F., 1982. The Insects: Structure and Function. Elsevier, New York. Deonier, D.L., 1971. A systematic and ecological study of nearctic Hydrellia (Diptera: Ephydridae). Smithsonian Contrib. Zool. 68, 10– 147. Doyle, R.D., Grodowitz, M.J., Smart, R.M., Owens, C., 2002. Impact of herbivory by Hydrellia pakistanae (Diptera: Ephydridae) on growth and photosynthetic potential of Hydrilla verticillata. Biol. Control 24, 221–229. Doyle, R., Grodowitz, M.J., Smart, M., Owens, C., 2007. Separate and interactive effects of competition and herbivory on the growth, expansion, and tuber formation of Hydrilla verticillata. Biol. Control. 41, 327–338. Freedman, J.E., Grodowitz, M.J., Cofrancesco, A.F., Bare, R., 2001. Mass-rearing Hydrellia pakistanae Deonier, A Biological Control Agent of Hydrilla verticillata (L.f.) Royle, for Release and Establishment. ERDC/EL TR-01-24, US Army Engineer Research and Development Center, Vicksburg, MS. Grodowitz, M.J., Freedman, J.E., Cofrancesco, A.F., Center, T.D., 1999. Status of Hydrellia spp. (Diptera: Ephydridae) Release Sites in Texas as of December 1998. Miscellaneous paper A-99-1, US Army Engineer Research and Development Center, Vicksburg, MS. Grodowitz, M.J., Freedman, J.E., Jones, H., Jeffers, L., Lopez, C., Nibling, F., 2000. Status of Waterhyacinth/Hydrilla Infestations and Associated Biological Control Agents in Lower Rio Grande Valley Cooperating Irrigation Districts, ERDC/EL SR-00-11, US Army Engineer Research and Development Center, Vicksburg, MS. Grodowitz, M.J., Smart, M., Doyle, R., Owens, C., Bare, R., Snell, C., Freedman, J., 2004. Hydrellia pakistanae and H. balciunasi, insect biocontrol agents of hydrilla: boon or bust?. In: Cullen, J.M., Briese, D.T., Kriticos, D.J., Lonsdale, W.M., Morin, L, Scott, J.K. (Eds.), Proceedings of XI International Symposium on Biological Control of Weeds; Apr 27–May 2 2003, Canberra, Australia, pp. 529–538. Hayes, E.J., Wall, R., 1999. Age-grading adult insects: a review of techniques. Physiol. Entomol. 24, 1–10. Tyndale-Biscoe, M., 1984. Age-grading methods in adult insects: a review. Bull. Entomol. Res. 74, 341–377. Van Geem, T., Broce, A.B., Moon, R.D., 1983. A System for Physiological Age-Grading of Female Face Flies Musca autumnalis De Geer. Bulletin 643. Agricultural Experiment Station, Kansas State University, Lawrence, KS. Wheeler, G.S., Center, T.D., 1996. The influence of hydrilla leaf quality on larval growth and development of the biological control agent Hydrellia pakistanae (Diptera: Ephydridae). Biol. Control 7, 1–9. Wheeler, G.S., Center, T.D., 2001. Impact of the biological control agent Hydrellia pakistanae (Diptera: Ephydridae) on the submersed aquatic weed Hydrilla verticillata (Hydrocharitaceae). Biol. Control 21, 168–181.
A physiological age-grading system for female Hydrellia pakistanae Deonier (Diptera: Ephydridae) Jennifer M. Lenz b
a,1
, Michael J. Grodowitz
b,*
, James H. Kennedy
a,1
a University of North Texas, Denton, TX 76203, USA US Army Engineer Research and Development Center, Vicksburg, MS 39180, USA
Received 5 September 2006; accepted 23 March 2007 Available online 24 April 2007
Abstract The female reproductive system of Hydrellia pakistanae Deonier was described by dissecting individuals of known ages and describing changes in relation to number of eggs oviposited. Female H. pakistanae have meroistic ovaries, in that specialized nurse cells or trophocytes (nutritive cells associated with the eggs) are present within each ovariole, and of the polytrophic subtype because nurse cells closely accompany each developing egg, or oocyte. The reproductive system includes two ovaries, each consisting of 8 or 10 tube-like ovarioles. The ovariole can be divided into two distinct areas: a distal germarium, which produces the follicles, and a more proximal vitellarium, which houses the developing follicles. Each ovariole is surrounded by an ovariole sheath and houses several developing follicles, a term referring to the ova (yolk and surrounding cells; i.e., the egg), nurse cells, and surrounding epithelium. Within an ovariole, the follicle proximate to the lateral oviduct is the most mature with each subsequent, more distal follicle becoming less mature. All ovarioles within an ovary are connected via the lateral oviducts, while the common oviduct connects the ovaries to each other. The spermatheca and accessory glands branch off the common oviduct. Sperm stored in the spermatheca fertilize eggs as they pass through the common oviduct. Based on this information the continuum of ovarian maturation was divided into three nulliparous and four parous stages. The nulliparous stages are classified based on degree of ovariole segmentation and maturity of the most proximal follicle while follicle relic quantity and appearance is mainly used to separate the parous classes. Limited field sampling demonstrated the possibility of using this system to assess reproductive health of wild populations. 2007 Elsevier Inc. All rights reserved. Keywords: Hydrellia pakistanae; Hydrilla verticillata; Biological control; Age-grading; Follicular relics
1. Introduction Hydrellia pakistanae Deonier, an introduced leaf-mining fly species in the family Ephydridae, is a potentially valuable organism for the biological control of Hydrilla verticillata (L.f.) Royle, an invasive aquatic plant. Native to Pakistan, India, and as far north as Beijing, China, H. pakistanae was first released in southern Florida in 1987. Currently, it has been released at over 50 sites in Alabama, *
Corresponding author. Fax: +1 601 634 2398. E-mail addresses: [email protected] (J.M. Lenz), [email protected] (M.J. Grodowitz), [email protected] (J.H. Kennedy). 1 Fax: +1 940 565 4297. 1049-9644/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2007.03.015
Arkansas, California, Florida, Georgia, Louisiana, North Carolina, Virginia, and Texas (Center et al., 1997). H. pakistanae is now established in Arkansas, Louisiana, Florida, Georgia, and Texas (Center et al., 1997) with range expansions noted in both Texas and Louisiana (Grodowitz et al., 2000; Julie Nachtrieb, US Army Engineer Lewisville Aquatic Ecosystem Research Facility, Lewisville, TX, personal communication and Jan Freedman, US Army Engineer Research and Development Center, Vicksburg, MS, personal communication. The larvae of H. pakistanae feed exclusively on hydrilla, and while they have been shown to impact photosynthesis and cause significant damage to experimental populations (Doyle et al., 2002, 2007), there are conflicting opinions as to their impact on field populations of hydrilla. H. pakistanae appears to be successful at
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controlling hydrilla in Texas field populations (Grodowitz et al., 1999); however, fly populations at many Florida sites have only achieved a maximum of 15% leaf damage to hydrilla, with many sites exhibiting much lower population levels (<200 immatures/kg) and associated damage (Wheeler and Center, 2001). Recent work by Grodowitz et al. (2004) has characterized the impact of the fly to both experimental and field populations. Reasons for such low population levels at many sites are unknown but several ideas have been cited. These include parasitism by a native pupal parasite (Trichopria columbiana Ashmead (Hymenoptera: Ichneumonidae) (Wheeler and Center, 2001; Doyle et al., 2002; Christie Snell and Robin Bare, US Army Engineer Lewisville Aquatic Ecosystem Research Facility, Lewisville, TX), plant nutritional changes influencing larval development (Wheeler and Center, 1996; Michael Grodowitz, Jan, Freedman, and Dwilette McFarland, unpublished), and high temperatures. While some of these ideas have been studied to an extent, one possible mechanism for low population development has been poorly addressed and only rarely mentioned as a possible mechanism. This is the impact adult nutrition has on reproductive development, egg production, and oviposition rates. Only minimal information is available on the reproductive development of H. pakistanae, let alone adult nutritional requirements. Most of the present literature focuses on its release, establishment, and success as a biological control agent. The first step toward adequately studying adult H. pakistanae nutritional requirements is to provide information describing, in detail, basic reproductive morphology in females as well as the overall development of the reproductive system through time. This is an important prerequisite to understanding the influence adult diet has on egg production and oviposition rates. Published accounts are of little help; only one paper describing the female reproductive system exists (Deonier, 1971) and only minimal morphological details are included in this paper. To address this gap in the literature, the research described here provides details of morphology of the female reproductive system and associated changes through time for H. pakistanae. It describes a physiological age-grading system based on changes in the female reproductive system. Such a system will be important for understanding and measuring changes in reproduction in relationship to adult nutritional requirements. 2. Materials and methods 2.1. Source of insects Rearing methods are modified only slightly from those found in the literature (Freedman et al., 2001). H. pakistanae larvae and adults were collected from colonies maintained at the Lewisville Aquatic Ecosystem Research Facility (LAERF) in Lewisville, Texas, and from colonies established in the Department of Biological Science at the
University of North Texas (UNT) in Denton, Texas. Both colonies consisted of immature rearing containers and oviposition boxes. Rearing containers were 4-l in volume and filled with about 75% hydrilla in water, leaving enough headspace for adults to mate and oviposit. Adults were periodically removed from the containers using an aspirator and sorted by sex and species. Those adults not used for experiments or dissections were placed into the oviposition box to encourage additional ovipositions. Oviposition boxes were slanted with a height measurement of 54.5 cm (back), 47 cm (front), and a depth of 63.5 cm. The front, measuring 122 cm across, had two circular openings allowing for internal access, each fitted with a cloth sleeve to prevent flies from escaping. The top of the oviposition box was constructed of Plexiglas to allow maximum light penetration. In each oviposition box, small petri dishes with several drops of two adult food mixtures and large petri dishes with hydrilla stems in water for use as an oviposition substrate were included. Immature larvae found on the hydrilla were placed back into the rearing containers to enlarge the colonies. Adult food consisted of a sugar–yeast hydrolyzate mixture (4 g yeast hydrolyzate, 7 g sucrose, 10 ml water) and a sugar–water mixture (1:1) (Freedman et al., 2001). Food was changed frequently to prevent formation of fungal contamination. Adults for field studies were collected from the surface of experimental ponds containing hydrilla at the LAERF using a hand-held, battery-powered insect vacuum. Flies were collected during the active growing period, i.e., June through September. 2.2. Dissection techniques Females were dissected alive using a stereomicroscope (25–35·) to prevent deterioration of internal organs that occurs shortly after death. Adults were held individually in plastic vials at room temperature until just prior to dissection. Adults were anesthetized by placing them briefly in a freezer (3–5 min) or by exposure to CO2. Inactive flies were removed and immediately pinned through the dorsal surface of the thorax; ventral side down, onto an electron microscopy stub covered with a mixture of beeswax, paraffin, and dark-colored crayon wax. The flies were covered completely with a phosphate buffered saline, pH 7 (PBS) in an effort to protect the ovaries from desiccation and deterioration due to osmotic changes. Grasping the abdominal cuticle along the mid-dorsal line with fine forceps and gently tearing the cuticle opened the abdominal cavity, exposing the ovaries. Prior to removal of the ovaries, characteristics of the fat body (if visible) were noted. Ovaries were carefully removed by grasping the common oviduct using fine forceps. The ovaries were subsequently placed onto a microscope slide in a drop of PBS for examination. The ovaries were first viewed using a dissecting microscope, and examined under a compound microscope at 100· and 200· magnifications to distinguish changes in reproductive system morphological markers. Characteris-
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tics such as the quantity and quality of follicular relics, number of ovarioles per ovary, number of mature ovarioles, and the condition of the ovaries and spermatheca were noted. Photomicrographs were taken at different magnifications under both dissecting and compound microscopes for later comparisons. 2.3. Baseline studies Hydrellia pakistanae females randomly collected from the rearing containers were used to practice dissection techniques and describe reproductive morphology. Chronological age, if known, was used to associate general reproductive changes over time. In order to associate physiological changes in the reproductive system with fecundity (number of eggs oviposited), females of a known age that had not yet oviposited were used. Two techniques for collecting young females were used. In one technique, young females (±0.50- to 0.75-h-old) were obtained from colonies by first removing all adults from the rearing containers. After 1 to 1.5 h, all newly emerged adults were collected. After this time, the females collected were not used, unless males were completely absent. While females often emerge with an almost fully mature reproductive system, no eggs are oviposited until mating is completed (personal observation). A second technique was developed to collect young females from field populations. Hydrilla stems with obvious fly damage were collected from experimental ponds at the LAERF. Pupae were removed (while still attached to the stem) and placed individually into vials with enough water to cover the stem. Since each pupa was isolated, it was not necessary to ensure the females were within 1 to 1.5 h old because no mating, and hence oviposition, could have occurred. H. pakistanae females collected via either technique were held in a plastic vial while a 240-ml holding container was readied. Each container held several short hydrilla stems in 30 to 60 ml water for oviposition, and a 1-cm2 floating piece of Styrofoam carrying one drop of each of the two adult food mixtures. The females were placed individually into the containers, and one or two males were added as soon as they were available. The containers were maintained in an incubator at 24–25 C with a photoperiod of 18 light: 6 dark and a relative humidity of about 50%. The containers were checked daily, and hydrilla stems removed and carefully inspected for eggs. Eggs were tallied and whorls containing eggs were removed to prevent re-counting. Each stem was checked twice if the female was about to be dissected. For females that were being held for a longer period, hydrilla stems without eggs were placed back into the cup so that any missed eggs could be enumerated at a later time. Fresh food was added and adults replaced. When larger numbers of eggs were needed, females were not handled for the first two days. This seemed to encourage additional ovipositions. Males were replaced if found dead because females apparently required their presence to continue ovipositing. If, upon
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dissection, an egg was found in the lateral or common oviduct, it was also counted since the epithelium and nurse cells that form relics had already been left behind. Dead females were dissected only if evidence indicated a recent death (within the hour). In this case, the data were only used if the ovaries showed no signs of deterioration and then only to corroborate other data in the same class. Researchers have shown (Buckingham and Okrah, 1993; Buckingham et al., 1989) that females feeding on the diet used in this study oviposited an average of 68 eggs. Hence, an attempt was made to observe females that had oviposited at least this many eggs. Females were observed that oviposited a total of 68 eggs (i.e., 3 or 4 full-batches depending on the number of ovarioles per ovary). It was difficult to get higher egg counts because it appeared that isolation and frequent handling caused females to lay fewer eggs. Although female H. pakistanae have been shown to oviposit >100 eggs under laboratory conditions (Buckingham and Okrah, 1993), it is believed that the appearance of the follicular relics at higher egg counts would not differ greatly from that seen in the highest class observed in this research. Space in the lateral oviduct for the relics to accumulate is limited, so after relics reach the size of clusters found in the highest class, they will most likely only become denser and darker. Approximately 200 H. pakistanae females were dissected for the baseline studies. Most of the flies were used to become familiar with the overall reproductive morphology and morphological changes that occur through time. More than 50 females were kept under the controlled conditions described earlier. The number of eggs oviposited and reproductive characteristics upon dissection were recorded for these females. These data were used to develop the physiological age-grading system by associating changes in the reproductive system with the number of eggs ovulated and oviposited. 2.4. Field studies The developed physiological age-grading system was tested on field populations at the LAERF experimental ponds. Flies were vacuumed from the surface of several ponds on five different days during June 2002. Collected Hydrellia spp. were identified to species by examining external genitalia. H. pakistanae females, distinguished by their L-shaped cerci, were dissected using the methods described above. The flies were placed into classes according to the developed physiological age-grading system. A total of 56 females were dissected. 3. Results and discussion 3.1. Reproductive morphology The H. pakistanae female reproductive system is as described by Deonier (1971) for Nearctic Hydrellia. It is meroistic, in that specialized nurse cells or trophocytes
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(nutritive cells associated with the eggs) are present within each ovariole, and of the polytrophic subtype because nurse cells closely accompany each developing egg or oocyte (Chapman, 1982). The reproductive system includes two ovaries, each consisting of 8 or 10 tube-like ovarioles (Fig. 1). The ovariole can be divided into two distinct areas: a distal germarium, which produces the follicles, and a more proximal vitellarium, which houses the developing follicles (Fig. 2a). Each ovariole is surrounded by an ovariole sheath and houses several developing follicles, a term referring to the ova (yolk and surrounding cells), nurse cells, and surrounding epithelium (Fig. 2b). Within an ovariole, the follicle proximate to the lateral oviduct is the most mature with each subsequent, more distal follicle becoming less mature. A mature follicle (one that is ready to be ovulated) appears full with yolk and has no visible nurse cells. An immature follicle has visible nurse cells with variations in yolk amount (Fig. 2b). All ovarioles within an ovary are connected via the lateral oviducts, while the common oviduct connects the ovaries to each other. The spermatheca and accessory glands branch off the common oviduct (Fig. 1). Sperm stored in the spermatheca fertilize eggs as they pass through the common oviduct. Maturation of proximate follicles (those closest to the lateral oviduct) appears to be independent between ovarioles. In other words, once a follicle is ovulated (i.e., passes to the lateral oviduct), the next distal follicle will begin to mature regardless of the maturity of the proximate follicles
in other ovarioles within the same ovary. Evidence of this is seen in ovaries that have proximate follicles of various states of maturity. For example, one ovary may have a single mature follicle as well as follicles of varying maturities, all of which are immediately distal to the lateral oviduct (Fig. 2c). Another example is when all the proximate follicles are immature, but one stands out as distinctly closer to maturity than the others (Fig. 2d). 3.2. Fat body The fat body is also used to determine physiological age. It is easily identified in the abdominal cavity and in recently emerged females the fat body appears diffuse and green (Fig. 3a and b) and becomes clustered and green within 1–2 h after emergence (Fig. 3c and d). The green color is apparently derived from pigments within hydrilla tissue consumed during the larval stages. Eventually the fat body changes color becoming milky white and minimal in quantity, making it difficult to observe. Changes in the fat body are only useful in distinguishing nulliparous classes (those where no eggs have been oviposited) and those classes with low egg counts because it does not change distinctly after oviposition begins. 3.3. Follicular relic formation As an oocyte passes through the lateral oviduct (i.e., ovulation), the surrounding epithelium and nurse cells slough off and accumulate within the base of the lateral
Fig. 1. Photomicrograph of the reproductive system of H. pakistanae showing the overall structure of each ovary, connecting oviducts, and associated glands. This example is from a female with eight ovarioles per ovary.
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Fig. 2. A composite plate with images depicting various morphological features of the H. pakistanae ovaries (a and b) as well as differences in follicle maturation (c and d). (a) Close-up of a single ovary from a female with 10 ovarioles per ovary showing the two distinct regions associated with follicles; i.e., the germarium (g) and vitellarium (v); (b) morphological features of a single immature follicle; (c) ovary having proximate follicles in various states of maturity—in this case there are even number of immature and mature proximate follicles as compared with plate (d), where most follicles are immature but one is distinctly more mature (d).
oviduct. These accumulations of cellular debris are known as follicular relics. As more and more cellular material is sloughed off in the lateral oviduct, the follicular relics become compressed and hence change in appearance, allowing for an estimation of fecundity. Two distinct types of relics are observed: brown follicular relics that are relatively loose in the ovariole sheath (Fig. 4a), and yellow follicular relics, which appear compressed and granular or particulate in the lateral oviducts (Fig. 4b). The brown follicular relics are presumably epithelium and nurse cells recently left behind after an ovulation, while yellow follicular relics are composed of the same
cellular remnants, only compressed, indicating that subsequent ovulations have taken place. Both types of relics may be found simultaneously. Characteristics of the yellow follicular relics can be used to estimate physiological age. Brown relics indicate a recent ovulation and can obscure the presence and appearance of the yellow relics. Yellow follicular relics are diffuse and scarce in females that have oviposited few eggs, but gradually become larger and more clustered with successive ovulations. The color of the yellow relics also appears darker with more ovulations due to the accumulation of material and compression of the relics. After an ovulation, the yellow relics can appear
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Fig. 3. Photomicrograph of diffuse green fat body seen in the abdominal cavity (a and b) contrasted with clustered fat body in (c and d).
Fig. 4. Photomicrograph of brown follicular relics (B) loose in the ovariole sheath with compressed yellow relics (Y) below (a). In (b), yellow follicular relics can be seen compressed in the lateral oviduct of an ovary.
particulate because they become broken apart by the passing oocyte. The volume in the lateral oviduct composed of yellow relics generally increases with a higher number of ovulations, but apparently is not reliable in determining fecundity. This occurs because some of the preexisting yellow relics are apparently flushed out of the lateral oviducts along with the egg during successive ovulations. Size of relics may also be reduced due to compression by passing fol-
licles. As evidence of this, no significant correlation (n = 45, P > 0.05, r = 0.259) between relic size and number of ovipositions was noted. 3.4. Physiological age descriptions Physiological age classes were based on changes in the morphology of the female reproductive system and allow for a rough estimation of the number of eggs oviposited
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(Fig. 5). Classes were identified using characteristics of both the fat body and follicular relics (Table 1). Two broad class types were noted: nulliparous classes (i.e., N1–N3), where no eggs have been oviposited, and parous classes (i.e., P1–P4), where eggs have been oviposited. 3.4.1. Nulliparous classes 1–3 (N1–N3) Females in these classes have deposited no eggs and the ovaries are distinguished by the absence of follicular relics. In N1, the fat body is highly diffuse and green, and takes up more than half of the abdominal cavity. None of the proximate follicles are mature (Fig. 6a). In Grodowitz et al. (1999) and Van Geem et al. (1983), the ovarioles in the N1 stage were described as being totally nondifferentiated; i.e., being without distinct follicles. This was never observed in H. pakistanae presumably because nondifferentiated ovarioles occur before adult eclosion and typically will not be observed under field conditions. In the N2 class, the fat body also occupies more than half of the abdominal cavity, but appears as distinct green clusters. Some of the proximate follicles are mature (Fig. 6b). N3 is the class just Fig. 6. (a) Photomicrograph of an ovary with no follicular relics and no mature follicles representing nulliparous class 1 or N1. (b) Photomicrograph of a pair of ovaries with no follicular relics and some mature follicles; i.e., three mature follicles in the right ovary and two in the left. This female represents nulliparous class 2 or N2 (b).
before ovulation occurs. In this class, the fat body is very similar to that of N2, but now most of the proximate follicles are fully mature and ready to be ovulated but no follicular relics are present.
Fig. 5. Mean number (±1 standard error and 95% confidence interval) of eggs oviposited by females in each parous class (P1–P4).
3.4.2. Parous class 1 (P1) Individuals in the P1 class have oviposited from one to about 15 eggs (Fig. 5). The fat body may still be present as distinct green clusters, but occupies less than half the abdominal cavity, or has become milky white and scarce. Proximate follicles may all be mature, all immature, or with some variable number of each. This type of variation is
Table 1 Physiological age classes and associated characteristics for H. pakistanae females Class
# eggs
Fat body
Ovariole condition
Follicular relics
N1 N2 N3
0 0 0
Dense, green Clustered, green Clustered, green
None mature Some mature All mature
None None None
P1
1–15
Scarce (color varies) or absent
None, some or all mature
Diffuse both ovaries, with or without brown relics Mostly diffuse with small clusters in one ovary, diffuse in other ovary
P2
15–40
Scarce (color varies) or absent
None, some or all mature
Diffuse with small clusters both ovaries Light particles both ovaries
P3
40–60
Scarce (color varies) or absent
None, some or all mature
Partly diffuse or particulate with large particulate clusters, one or both ovaries Large particulate clusters both ovaries
P4
60+
Scarce (color varies) or absent
None, some or all mature
Large dense clusters both ovaries
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seen in all parous classes. It is possible to observe the presence of only brown relics (indicating one or two ovulations) but care must be taken not to miss yellow relics, which can be easily obscured by the brown relics. Relics in both ovaries are scarce, very light, and diffuse, with no brown relics present in the earlier stages of this class (Fig. 7a), or the same accompanied by brown relics towards the middle of
the class (Fig. 7b). In late P1 individuals, yellow relics are still mostly diffuse, but start to appear clustered in one ovary indicating subsequent ovipositions (Fig. 7c). 3.4.3. Parous class 2 (P2) Oviposition number ranges from the mid-teens to <40 for individuals in the P2 class (Fig. 5). The fat body in this
Fig. 7. Series of photomicrographs depicting the various parous classes. The first series of images (a, b, and c) represents parous class 1 (P1) as indicated by minimal quantities of diffuse follicular relics. The number of eggs oviposited by these three different females ranges from 3 to 14. Photomicrographs (d and e) show mostly diffuse yellow relics with only small clusters in both ovaries. These females oviposited from 16 to 35 eggs and belong in parous class 2 (P2). Photomicrographs in (f, g, and h) all show diffuse yellow relics with large particulate clusters in both ovaries. These females oviposited from 44 to 56 eggs and belong in parous class 3 (P3). Photomicrograph of a pair of ovaries showing large dense yellow relics in both (i). This female laid 62 eggs and belongs in parous class 4 or P4.
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class (and subsequent classes) is usually very scarce and may easily be missed. Yellow relics are still mostly diffuse, but begin to appear clustered in both ovaries and may or may not be accompanied by brown relics (Fig. 7d and e). It is possible for relics in one ovary to be light and diffuse, with the other ovary having relics that show signs of recent ovipositions, i.e., brown relics and particulate yellow relics. This is only seen in the earlier stages of this class. Both ovaries may also have yellow relics that appear particulate. 3.4.4. Parous class 3 (P3) Individuals in the P3 class have an oviposition range >39 and <60 (Fig. 5). In this class, the yellow relics appear as large particulate clusters, but are usually partially diffuse or particulate (Fig. 7f, g, and h). Occasionally, both ovaries have particulate yellow relics, where the particles are medium in color and take up a large amount of space in the lateral oviduct. This amount of space is about equal to the size of the yolk in a half-mature follicle (where the yolk equals half the total size of the follicle). 3.4.5. Parous class 4 (P4) This class contains individuals with ovipositions P60. The yellow relics begin to appear as large dense clusters. The dense clusters resemble the clusters in P2, but are larger and darker (Fig. 7i). Size of the clusters is comparable to the size of the circular nurse cells seen in immature follicles or it may be larger. This physiological age-grading system provides a rough approximation of ovipositions and is designed to distinguish females that have not oviposited or have laid only a few eggs from those that have oviposited higher numbers (Fig. 5). There is some degree of overlap of the characteristics found in each class and as ovipositions increase, variation of the characteristics associated with each class increases. For this reason, this system will provide only an estimate, but not an exact number, of oviposition numbers for field populations. 3.5. Field studies Females examined from experimental field populations at LAERF demonstrate application of the developed system. H. pakistanae females could easily be assigned to the described classes and the observed characteristics did not differ from those in the controlled baseline studies. No females were nulliparous, but this is not surprising because females will mate and oviposit within 1–1.5 h after emerging. Since the nulliparous classes apparently last for only a short time, individuals in these classes may be difficult to observe in field populations. Also, females that have recently emerged may not be as active and thus, not as likely to be collected. The lack of individuals in the nulliparous classes can be expected because it is typical to have under-representation in the youngest age classes (Hayes and Wall, 1999). Approximately 15% of the dissected field flies were classified as P1 (Fig. 8). Larger proportions (50%)
Fig. 8. Percent of total number of female H. pakistanae from LAERF experimental field populations in each parous class. The column between P3 and P4 represents those with characteristics of both classes.
of the flies were classified as P2. Since this class is rather large, and encompasses the middle of the physiological age distribution, this was not unexpected. Approximately 20% were classified as P3, while another 9% showed characteristics of both P3 and P4. These females most likely had oviposition numbers in the high 50 s or low 60 s. The remaining 7% of the females were classified as P4. This means that 7% of H. pakistanae females from the population sampled each laid 60 or more eggs; i.e., 3 or 4 full-complements (depending on the number of ovarioles per ovary). 3.6. Importance of physiological age-grading systems Physiological age-grading systems are useful, particularly for insects, where physiological (rather than chronological) age determines many characteristics, such as fecundity, certain behaviors, and lifespan (Tyndale-Biscoe, 1984). In the area of biological control, physiological agegrading systems allow researchers to estimate the future impact of a release, as well as to ascertain the success of previous releases. For example, successful biological control agents must be able to reproduce and buildup large populations to control the target pest. This can be assessed fairly easily using such a developed system. Physiological age-grading systems can also be used to track reproductive status of the biological control agent through time. This can provide helpful information such as peak reproductive activity of the released individuals. Determining physiological, rather than chronological, age is valuable because the two may differ considerably. While chronological age is solely based on time and is not influenced by outside factors, physiological age is based on number of eggs oviposited and is influenced by environmental factors such as climate and nutritional supply. Creating a physiological age-grading system for H. pakistanae using ovarian morphology to evaluate reproductive
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status of the fly in field populations will be useful in assessing impacts of H. pakistanae on hydrilla and measure the potential success of releases based on reproductive activity of the introduced individuals. Knowing the relative numbers of eggs being oviposited by females provides researchers an idea of the physiological age distribution of the fly. This information is useful in estimating size of future generations, and therefore possible impact on hydrilla. Although, the exact number of ovipositions cannot be determined, researchers can easily distinguish flies that have oviposited relatively few eggs from those that have oviposited many. This information will provide some evidence as to whether or not H. pakistanae are depositing enough eggs to maintain populations sufficient to control hydrilla. It will also give important clues to the varying success of H. pakistanae as a biological control agent for hydrilla. Acknowledgments This research was accomplished as part of the requirement for completion of a Masters Thesis of the senior author at the University of North Texas in Denton, Texas. The US Army Engineer Research and Development Center provided funding for this project under the Aquatic Plant Control Research Program, Dr. John Barko, Program Manager. The Lewisville Aquatic Ecosystem Research Facility in Lewisville, Texas provided use of experimental ponds, fly colony, and equipment. Thanks to Mses. Robin Bare, Christi Snell, Chetta Owens, and Jessica Sharp for technical assistance. Thanks are also extended to the author’s Thesis committee members, Drs. Ken Dickson and Tom Waller. References Buckingham, G.R., Okrah, E.A., 1993. Biological and Host Range Studies with Two Species of Hydrellia (Diptera: Ephydridae) that Feed on Hydrilla. Technical Report A-93-7, US Army Engineer Waterways Experiment Station, Vicksburg, MS. Buckingham, G.R., Okrah, E.A., Thomas, M.C., 1989. Laboratory host range tests with Hydrellia pakistanae (Diptera: Ephydridae), an agent for biological control of Hydrilla verticillata (Hydrocharitaceae). Environ. Entomol. 16, 164–171. Center, T.D., Grodowitz, M.J., Cofrancesco, A.F., Jubinsky, G., Snoddy, E., Freedman, J.E., 1997. Establishment of Hydrellia pakistanae (Diptera: Ephydridae) for the biological control of the submersed
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